Tendon-inducing compositions

ABSTRACT

Compositions of proteins with tendon/ligament-like tissue inducing activity are disclosed. The compositions are useful in the treatment of tendinitis and tendon or ligament defects and in related tissue repair.

RELATED APPLICATIONS

The present invention is a continuation-in-part of application Ser. No.08/217,780, filed Mar. 25, 1994, Ser. No. 08/164,103, filed on Dec. 7,1993 and Ser. No. 08/333,576, filed on Nov. 2, 1994.

FIELD OF THE INVENTION

The present invention relates to a novel family of purified proteins,and compositions containing such proteins, which compositions are usefulfor the induction of tendon/ligament-like tissue formation, woundhealing and ligament and other tissue repair. These proteins may also beused in compositions for augmenting the activity of bone morphogeneticproteins.

BACKGROUND OF THE INVENTION

The search for the molecule or molecules responsible for formation ofbone, cartilage, tendon and other tissues present in bone and othertissue extracts has led to the discovery of a novel set of moleculescalled the Bone Morphogenetic Proteins (BMPs). The structures of severalproteins, designated BMP-1 through BMP-11, have previously beenelucidated. The unique inductive activities of these proteins, alongwith their presence in bone, suggests that they are important regulatorsof bone repair processes, and may be involved in the normal maintenanceof bone tissue. There is a need to identify additional proteins whichplay a role in forming other vital tissues. The present inventionrelates to the identification of a family of proteins, which havetendon/ligament-like tissue inducing activity, and which are useful incompositions for the induction of tendon/ligament-like tissue formationand repair.

SUMMARY OF THE INVENTION

In one embodiment, the present invention comprises DNA moleculesencoding a tendon/ligament-like inducing protein which the inventorshave named V1-1. This novel protein is now called BMP-12. The presentinvention also includes DNA molecules encoding BMP-12 related proteins.BMP-12 related proteins are a subset of the BMP/TGF-β/Vg-1 family ofproteins, including BMP-12 and VL-1, which are defined astendon/ligament-like tissue inducing proteins encoded by DNA sequenceswhich are cloned and identified, e.g., using PCR, using BMP-12 specificprimers, such as primers #6 and #7 described below, with reducedstringency conditions. It is preferred that the DNA sequences encodingBMP-12 related proteins share at least about 80% homology at the aminoacid level from amino acids with amino acids #3 to #103 of SEQ ID NO:1.

The DNA molecules preferably have a DNA sequence encoding the BMP-12protein, the sequence of which is provided in SEQ ID NO:1, or a BMP-12related protein as further described herein. Both the BMP-12 protein andBMP-12 related proteins are characterized by the ability to induce theformation of tendon/ligament-like tissue in the assay described in theexamples.

The DNA molecules of the invention preferably comprise a DNA sequence,as described in SEQUENCE ID NO:1; more preferably nucleotides #496 to#882, #571 to #882 or #577 to #882 of SEQ ID NO:1; or DNA sequenceswhich hybridize to the above under stringent hybridization conditionsand encode a protein which exhibits the ability to formtendon/ligament-like tissue. The DNA molecules of the invention may alsocomprise a DNA sequence as described in SEQ ID NO:25; more preferablynucleotides #604 or #658 to #964 of SEQ ID NO:25.

The DNA molecules of the invention also include DNA molecules comprisinga DNA sequence encoding a BMP-12 related protein with the amino acidsequence shown in SEQ ID NO:2 or SEQ ID NO:26, as well as naturallyoccurring allelic sequences and equivalent degenerative codon sequencesof SEQ ID NO:2 or SEQ ID NO:26. Preferably, the DNA sequence of thepresent invention encodes amino acids #-25 to # 104, #1 to # 104 or #3to #103 of SEQ ID NO:2; or amino acids #1 to #120 or #19 to #120 of SEQID NO:26. The DNA sequence may comprise, in a 5′ to 3′ direction,nucleotides encoding a propeptide, and nucleotides encoding for aminoacids #-25 to #104, #1 to #104 or #3 to #103 of SEQ ID NO:2; or aminoacids #1 to #120 or #19 to #120 of SEQ ID NO:26. The propeptide usefulin the above embodiment is preferably selected from the group consistingof native BMP-12 propeptide and a protein propeptide from a differentmember of the TGF-B superfamily or BMP family. The invention furthercomprises DNA sequences which hybridize to the above DNA sequences understringent hybridization conditions and encode a BMP-12 related proteinwhich exhibits the ability to induce formation of tendon/ligament-liketissue.

In other embodiments, the present invention comprises host cells andvectors which comprise a DNA molecule encoding the BMP-12 protein, or aBMP-12 related protein. The host cells and vectors may further comprisethe coding sequence in operative association with an expression controlsequence therefor.

In another embodiment, the present invention comprises a method forproducing a purified BMP-12 related protein, said method comprising thesteps of culturing a host cell transformed with the above DNA moleculeor vector comprising a nucleotide sequence encoding a BMP-12 relatedprotein; and (b) recovering and purifying said BMP-12 related proteinfrom the culture medium. In a preferred embodiment, the method comprises(a) culturing a cell transformed with a DNA molecule comprising thenucleotide sequence from nucleotide #496, #571 or #577 to #879 or #882as shown in SEQ ID NO:1; or the nucleotide sequence from #604 or #658 to#963 of SEQ ID NO:25; and

(b) recovering and purifying from said culture medium a proteincomprising the amino acid sequence from amino acid #-25, #1 or #3 toamino acid #103 or #104 as shown in SEQ ID NO:2; or from amino acid #1or #19 to amino acid #120 as shown in SEQ ID NO:26. The presentinvention also includes a purified protein produced by the abovemethods.

The present invention further comprises purified BMP-12 related proteincharacterized by the ability to induce the formation oftendon/ligament-like tissue. The BMP-12 related polypeptides preferablycomprise an amino acid sequence as shown in SEQ ID NO:2. The polypeptidemore preferably comprise amino acids #-25, #1 or #3 to #103 or #104 asset forth in SEQ ID NO:2; or amino acids #1 or #19 to #120 as set forthin SEQ ID NO:26. In a preferred embodiment, the purified polypeptide maybe in the form of a dimer comprised of two subunits, each with the aminoacid sequence of SEQ ID NO:2.

In another embodiment, the present invention comprises compositionscomprising an effective amount of the above-described BMP-12 relatedproteins. In the compositions, the protein may be admixed with apharmaceutically acceptable vehicle.

The invention also includes methods for tendon/ligament-like tissuehealing and tissue repair, for treating tendinitis, or other tendon orligament defects, and for inducing tendon/ligament-like tissue formationin a patient in need of same, comprising administering to said patientan effective amount of the above composition.

Other embodiments include chimeric DNA molecules comprising a DNAsequence encoding a propeptide from a member of the TGF-β superfamily ofproteins linked in correct reading frame to a DNA sequence encoding aBMP-12 related polypeptide. One suitable propeptide is the propeptidefrom BMP-2. The invention also includes heterodimeric protein moleculescomprising one monomer having the amino acid sequence shown in SEQ IDNO:2, and one monomer having the amino acid sequence of another proteinof the TGF-62 subfamily.

Finally, the present invention comprises methods for inducingtendon/ligament-like tissue formation in a patient in need of samecomprising administering to said patient an effective amount of acomposition comprising a protein which exhibits the ability to induceformation of tendon/ligament-like tissue, said protein having an aminoacid sequence shown in SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:26. Theamino acid sequences are more preferably one of the following: (a) aminoacids #-25, #1 or #3 to #103 or #104 of SEQ ID NO:2; (b) amino acids #1or #19 to #119 or #120 of SEQ ID NO:4; (c) amino acids #1 or #19 to #119or #120 of SEQ ID NO:26; (d) mutants and/or variants of (a), (b) or (c)which exhibit the ability to form tendon and/or ligament. In otherembodiments of the above method, the protein is encoded by a DNAsequence of SEQ ID NO: 1, SEQ ID NO:3 or SEQ ID NO:25, more preferablyone of the following: (a) nucleotides #496, #571 or #577 to #879 or #882of SEQ ID NO:1; (b) nucleotides #845 or #899 to #1201 or #1204 of SEQ IDNO:3; (c) nucleotides #605 or #659 to #961 or #964 of SEQ ID NO:25; and(d) sequences which hybridize to (a) or (b) under stringenthybridization conditions and encode a protein which exhibits the abilityto form tendon/ligament-like tissue.

DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is the nucleotide sequence encoding the human BMP-12.

SEQ ID NO:2 is the amino acid sequence comprising the mature humanBMP-12 polypeptide.

SEQ ID NO:3 is the nucleotide sequence encoding the protein MP52.

SEQ ID NO:4 is the amino acid sequence comprising the mature MP52polypeptide.

SEQ ID NO:5 is the nucleotide sequence of a specifically amplifiedportion of the human BMP-12 encoding sequence.

SEQ ID NO:6 is the amino acid sequence encoded by the nucleotidesequence of SEQ ID NO:5.

SEQ ID NO:7 is the nucleotide sequence of a specifically amplifiedportion of the human VL-1 encoding sequence.

SEQ ID NO:8 is the amino acid sequence encoded by the nucleotidesequence of SEQ ID NO:7.

SEQ ID NO:9 is the nucleotide sequence of the plasmid pALV1-781, usedfor expression of BMP-12 in E. coli.

SEQ ID NO:10 is the nucleotide sequence of a fragment of the murineclone, mV1.

SEQ ID NO:11 is the amino acid sequence of a fragment of the murineprotein encoded by mV1.

SEQ ID NO:12 is the nucleotide sequence of a fragment of the murineclone, mV2.

SEQ ID NO:13 is the amino acid sequence of a fragment of the murineprotein encoded by mV2.

SEQ ID NO:14 is the nucleotide sequence of a fragment of the murineclone, mV9.

SEQ ID NO:15 is the amino acid sequence of a fragment of the murineprotein encoded by mV9.

SEQ ID NO:16 is the amino acid sequence of a BMP/TGF-β/Vg-1 proteinconsensus sequence. The first Xaa represents either Gln or Asn; thesecond Xaa represents either Val or Ile.

SEQ ID NO:17 is the nucleotide sequence of oligonucleotide #1.

SEQ ID NO:18 is the amino acid sequence of a BMP/TGF-β/Vg-1 proteinconsensus sequence. The Xaa represents either Val or Leu.

SEQ ID NO:19 is the nucleotide-sequence of oligonucleotide #2.

SEQ ID NO:20 is the nucleotide sequence of oligonucleotide #3.

SEQ ID NO:21 is the nucleotide sequence of oligonucleotide #4.

SEQ ID NO:22 is the nucleotide sequence of oligonucleotide #5

SEQ ID NO:23 is the nucleotide sequence of oligonucleotide #6.

SEQ ID NO:24 is the nucleotide sequence of oligonucleotide #7.

SEQ ID NO:25 is the nucleotide sequence of the human VL-1 (BMP-13)encoding sequence.

SEQ ID NO:26 is the amino acid sequence encoded by the nucleotidesequence of SEQ ID NO:25.

SEQ ID NO:27 is the nucleotide sequence encoding a fusion of BMP-2propeptide and the mature coding sequence of BMP-12.

SEQ ID NO:28 is the amino acid sequence encoded by the nucleotidesequence of SEQ ID NO:27.

SEQ ID NO:29 is the nucleotide sequence encoding the murine mV1 protein.X01 is Val, Ala, Glu or Gly; X02 is Ser, Pro Thr or Ala; X03 is Ser orArg; X04 is Leu, Pro, Gln or Arg; X05 is Cys or Trp; X06 is Val, Ala,Asp or Gly; X07 is Val, Ala, Glu or Gly; X08 is Gln, Lys or Glu.

SEQ ID NO:30 is the amino acid sequence encoded by the nucleotidesequence of SEQ ID NO:29. X01 through X08 are the same as in SEQ ID NO:29.

SEQ ID NO:31 is the nucleotide sequence encoding the murine mV2 protein.X01 is Pro or Thr; X02 is Val.

SEQ ID NO:32 is the amino acid sequence encoded by the nucleotidesequence of SEQ ID NO:31. X01 and X02 are the same as in SEQ ID NO:31.

SEQ ID NO:33 is the nucleotide sequence encoding human BMP-12 protein.

SEQ ID NO:34 is the amino acid sequence encoded by the nucleotidesequence of SEQ ID NO:33.

SEQ ID NO:35 is the nucleotide sequence of oligonucleotide #8.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a comparison of the human BMP-12 and human MP52 sequences.

DETAILED DESCRIPTION OF THE INVENTION

The DNA sequences of the present invention are useful for producingproteins which induce the formation of tendon/ ligament-like tissue, asdescribed further below. The DNA sequences of the present invention arefurther useful for isolating and cloning further DNA sequences encodingBMP-12 related proteins with similar activity. These BMP-12 relatedproteins may be homologues from other species, or may be relatedproteins within the same species.

Still, a further aspect of the invention are DNA sequences coding forexpression of a tendon/ligament-like tissue inducing protein. Suchsequences include the sequence of nucleotides in a 5′ to 3′ directionillustrated in SEQ ID NO:1 or SEQ ID NO:25, DNA sequences which, but forthe degeneracy of the genetic code, are identical to the DNA sequenceSEQ ID NO:1 or 25, and encode the protein of SEQ ID NO:2 or 26. Furtherincluded in the present invention are DNA sequences which hybridizeunder stringent conditions with the DNA sequence of SEQ ID NO:1 or 25and encode a protein having the ability to induce the formation oftendon or ligament. Preferred DNA sequences include those whichhybridize under stringent conditions as described in Maniatis et al,Molecular Cloning (A Laboratory Manual), Cold Spring Harbor Laboratory(1982), pages 387 to 389. Finally, allelic or other variations of thesequences of SEQ ID NO:1 or 25, whether such nucleotide changes resultin changes in the peptide sequence or not, but where the peptidesequence still has tendon/ligament-like tissue inducing activity, arealso included in the present invention.

The human BMP-12 DNA sequence (SEQ ID NO:1) and amino acid sequence (SEQID NO:2) are set forth in the Sequence Listings. Another protein that isuseful for the compositions and methods of the present invention isVL-1. VL-1 is a BMP-12 related protein which was cloned using sequencesfrom BMP-12. The inventors have now designated VL-1 as BMP-13. A partialDNA sequence of VL-1 (SEQ ID NO:7) and the encoded amino acid sequence(SEQ ID NO:8); as well as a DNA sequence encoding the mature VL-1 (SEQID NO:25) and the encoded amino acid sequence (SEQ ID NO:26) are setforth in the Sequence Listings. Although further descriptions are madewith reference to the BMP-12 sequence of SEQ ID NO:1 and 2, it will berecognized that the invention includes similar modifications andimprovements which may be made to other BMP-12 related sequences, suchas the VL-1 sequence shown in SEQ ID NO:25 and 26.

The sequence of BMP-12 shown in SEQ ID NO.1 includes the entire maturesequence and approximately 190 amino acids of the propeptide. The codingsequence of the mature human BMP-12 protein appears to begin atnucleotide #496 or #571 and continues through nucleotide #882 of SEQ IDNO:1. The first cysteine in the seven cysteine structure characteristicof TGF-β proteins begins at nucleotide #577. The last cysteine ends at#879. Thus, it is expected that DNA sequences encoding active BMP-12species will comprise nucleotides #577 to #879 of SEQ ID NO:1.

It is expected that BMP-12, as expressed by mammalian cells such as CHOcells, exists as a heterogeneous-population of active species of BMP-12protein with varying N-termini. It is expected that all active specieswill contain the amino acid sequence beginning with the cysteine residueat amino acid #3 of SEQ ID NO:2 and continue through at least thecysteine residue at amino acid 103 or until the stop codon after aminoacid 104. Other active species contain additional amino acid sequence inthe N-terminal direction. As described further herein, the N-termini ofactive species produced by mammalian cells are expected to begin afterthe occurrence of a consensus cleavage site, encoding a peptide sequenceArg-X-X-Arg. Thus, it is expected that DNA sequences encoding activeBMP-12 proteins will have a nucleotide sequence comprising thenucleotide sequence beginning at any of nucleotides #196, 199, 208, 217,361, 388, 493, 496 or 571 to nucleotide #879 or 882 of SEQ ID NO:1.

The N-terminus of one active species of human BMP-12 has beenexperimentally determined by expression in E. coli to be as follows:[M]SRXSRKPLHVDF, wherein X designates an amino acid residue with noclear signal, which is consistent with a cysteine residue at thatlocation. Thus, it appears that the N-terminus of this species of BMP-12is at amino acid #1 of SEQ ID NO:1, and a DNA sequence encoding saidspecies of BMP-12 would start at nucleotide #571 of SEQ ID NO:1. Theapparent molecular weight of this species of human BMP-12 dimer wasdetermined by SDS-PAGE to be approximately 20-22 kd on a Novex 16%tricine gel. The pI of this molecule is approximately 4.9. The humanBMP-12 protein exists as a clear, colorless solution in 0.1%trifluoroacetic acid. The N-terminus of another active species of humanBMP-12 has been experimentally determined by expression in E. coli to be[M]TALA. The pI of this molecule is approximately 7.0. The apparentmolecular weight of this species of human BMP-12 dimer was determined bySDS-PAGE to be approximately 25-27 kd on a Novex 16% tricine gel. Thehuman BMP-12 protein exists as a clear, colorless solution in 0. 1%trifluoroacetic acid.

As described earlier, BMP-12 related proteins are a subset of theBMP/TGF-β/Vg-1 family of proteins, including BMP-12 and VL-1, which canbe defined as tendon/ligament-like tissue inducing proteins encoded byDNA sequences which can be cloned and identified, e.g., using PCR, usingBMP-12 specific primers, such as primers #6 and #7 described below, withreduced stringency conditions. It is preferred that DNA sequences of thepresent invention share at least about 80% homology at the amino acidlevel from amino acids with the DNA encoding amino acids #3 to #103 ofSEQ ID NO:1. For the purposes of the present invention, the term BMP-12related proteins does not include the human MP52 protein. Using thesequence information of SEQ ID NO:1 and SEQ ID NO:3, and the comparisonprovided in FIG. 1, it is within the skill of the art to design primersto the BMP-12 sequence which will allow for the cloning of genesencoding BMP-12 related proteins.

One example of the BMP-12-related proteins of the present invention isVL-1, presently referred to as BMP-13. The sequence of the full matureBMP-13 sequence and at least a part of the propeptide of BMP-13 is givenin SEQ ID NO:25. Like BMP-12, it is expected that BMP-13, as expressedby mammalian cells such as CHO cells, exists as a heterogeneouspopulation of active species of BMP-13 protein with varying N-termini.It is expected that all active species will contain the amino acidsequence beginning with the cysteine residue at amino acid #19 of SEQ IDNO:26 and continue through at least the cysteine residue at amino acid119 or until the stop codon after amino acid 120. Other active speciescontain additional amino acid sequence in the N-terminal direction. Asdescribed further herein, the N-termini of active species produced bymammalian cells are expected to begin after the occurrence of aconsensus cleavage site, encoding a peptide sequence Arg-X-X-Arg. Thus,it is expected that DNA sequences encoding active BMP-13 proteins willhave a nucleotide sequence comprising the nucleotide sequence beginningat any of nucleotides #410, 458, 602, 605 or 659, to nucleotide #961 or964 of SEQ ID NO:25.

In order to produce the purified tendon/ligament-like tissue inducingproteins useful for the present invention, a method is employedcomprising culturing a host cell transformed with a DNA sequencecomprising a suitable coding sequence, particularly the DNA codingsequence from nucleotide #496, #571 or # 577 to #879 or #882 of SEQ IDNO:1; and recovering and purifying from the culture medium a proteinwhich contains the amino acid sequence or a substantially homologoussequence as represented by amino acids #-25, #1 or #3 to #103 or #104 ofSEQ ID NO:2. In another embodiment, the method employed comprisesculturing a host cell transformed with a DNA sequence comprising asuitable coding sequence, particularly the DNA coding sequence fromnucleotide #605 or # 659 to #961 or #964 of SEQ ID NO:25; and recoveringand purifying from the culture medium a protein which contains the aminoacid sequence or a substantially homologous sequence as represented byamino acids #1 or #19 to #119 or #120 of SEQ ID NO:26.

The human MP52 DNA is described in WO93/16099, the disclosure of whichis incorporated herein by reference. However, this document does notdisclose the ability of the protein to form tendon/ligament-like tissue,or its use in compositions for induction of tendon/ligament-like tissue.Human MP52 was originally isolated using RNA from human embryo tissue.The human MP52 nucleotide sequence (SEQ ID NO:3) and the encoded aminoacid sequences (SEQ ID NO:4) are set forth in the Sequence Listingsherein. The MP52 protein appears to begin at nucleotide #845 of SEQ IDNO:3 and continues through nucleotide #1204 of SEQ ID NO:3. The firstcysteine of the seven cysteine structure characteristic of TGF-βproteins begins at nucleotide #899. The last cysteine ends at #1201.Other active species of MP52 protein may have additional nucleotides atthe N-terminal direction from nucleotide #845 of SEQ ID NO:3.

Purified human MP52 proteins of the present invention may be produced byculturing a host cell transformed with a DNA sequence comprising the DNAcoding sequence of SEQ ID NO:3 from nucleotide #845 to #1204, andrecovering and purifying from the culture medium a protein whichcontains the amino acid sequence or a substantially homologous sequenceas represented by amino acids #1 to #120 of SEQ ID NO:4. It is alsoexpected that the amino acid sequence from amino acids #17 or #19 to#119 or #120 of SEQ ID NO:4 will retain activity. Thus, the DNA sequencefrom nucleotides #845, #893 or #899 to #1201 or #1204 are expected toencode active proteins.

For expression of the protein in mammalian host cells, the host cell istransformed with a coding sequence encoding a propeptide suitable forthe secretion of proteins by the host cell is linked in proper readingframe to the coding sequence for the mature protein. For example, seeU.S. Pat. No. 5,168,050, the disclosure of which is hereby incorporatedby reference, in which a DNA encoding a precursor portion of a mammalianprotein other than BMP-2 is fused to the DNA encoding a mature BMP-2protein. Thus, the present invention includes chimeric DNA moleculescomprising a DNA sequence encoding a propeptide from a member of theTGF-β superfamily of proteins, is linked in correct reading frame to aDNA sequence encoding a tendon/ligament-like tissue inducingpolypeptide. The term “chimeric” is used to signify that the propeptideoriginates from a different polypeptide than the encoded maturepolypeptide. Of course, the host cell may be transformed with a DNAsequence coding sequence encoding the native propeptide linked incorrect reading frame to a coding sequence encoding the mature proteinshown in SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:26. The full sequence ofthe native propeptide may be determined through methods known in the artusing the sequences disclosed in SEQ ID NO:1, SEQ ID NO:3, or SEQ IDNO:25 to design a suitable probe for identifying and isolating theentire clone.

The present invention also encompasses the novel DNA sequences, free ofassociation with DNA sequences encoding other proteinaceous materials,and coding for expression of tendon/ligament-like tissue inducingproteins. These DNA sequences include those depicted in SEQ ID NO:1 in a5′ to 3′ direction and those sequences which hybridize thereto understringent hybridization conditions [for example, 0.1×SSC, 0.1% SDS at65° C.; see, T. Maniatis et al, Molecular Cloning (A Laboratory Manual),Cold Spring Harbor Laboratory (1982), pages 387 to 389] and encode aprotein having tendon/ligament-like tissue inducing activity.

Similarly, DNA sequences which code for proteins coded for by thesequences of SEQ ID NO:1 or SEQ ID NO:25, or proteins which comprise theamino acid sequence of SEQ ID NO:2 or SEQ ID NO:26, but which differ incodon sequence due to the degeneracies of the genetic code or allelicvariations (naturally-occurring base changes in the species populationwhich may or may not result in an amino acid change) also encode thetendon/ligament-like tissue inducing proteins described herein.Variations in the DNA sequences of SEQ ID NO:1 or SEQ ID NO:25 which arecaused by point mutations or by induced modifications (includinginsertion, deletion, and substitution) to enhance the activity,half-life or production of the polypeptides encoded are also encompassedin the invention.

Another aspect of the present invention provides a novel method forproducing tendon/ligament-like tissue inducing proteins. The method ofthe present invention involves culturing a suitable cell line, which hasbeen transformed with a DNA sequence encoding a protein of theinvention, under the control of known regulatory sequences. Thetransformed host cells are cultured and the proteins recovered andpurified from the culture medium. The purified proteins aresubstantially free from other proteins with which they are co-producedas well as from other contaminants.

Suitable cells or cell lines may be mammalian cells, such as Chinesehamster ovary cells (CHO). As described above, expression of protein inmammalian cells requires an appropriate propeptide to assure secretionof the protein. The selection of suitable mammalian host cells andmethods for transformation, culture, amplification, screening, productproduction and purification are known in the art. See, e.g., Gething andSambrook, Nature, 293:620-625 (1981), or alternatively, Kaufman et al,Mol. Cell. Biol., 5(7):1750-1759 (1985) or Howley et al, U.S. Pat. No.4,419,446. Another suitable mammalian cell line, which is described inthe accompanying examples, is the monkey COS-1 cell line. The mammaliancell CV-1 may also be suitable.

Bacterial cells may also be suitable hosts. For example, the variousstrains of E. coli (e.g., HB101, MC1061) are well-known as host cells inthe field of biotechnology. Various strains of B. subtilis, Pseudomonas,other bacilli and the like may also be employed in this method. Forexpression of the protein in bacterial cells, DNA encoding a propeptideis not necessary.

Bacterial expression of mammalian proteins, including members of theTGF-β family is known to produce the proteins in a non-glycosylatedform, and in the form of insoluble pellets, known as inclusion bodies.Techniques have been described in the art for solubilizing theseinclusion bodies, denaturing the protein using a chaotropic agent, andrefolding the protein sufficiently correctly to allow for theirproduction in a soluble form. For example, see EP 0433225, thedisclosure of which is hereby incorporated by reference.

Alternatively, methods have been devised which circumvent inclusion bodyformation, such as expression of gene fusion proteins, wherein thedesired protein is expressed as a fusion protein with a fusion partner.The fusion protein is later subjected to cleavage to produce the desiredprotein. One example of such a gene fusion expression system for E. coliis based on use of the E. coli thioredoxin gene as a fusion partner,LaVallie et al., Bio/Technology, 11:187-193 (1993), the disclosure ofwhich is hereby incorporated by reference.

Many strains of yeast cells known to those skilled in the art may alsobe available as host cells for expression of the polypeptides of thepresent invention. Additionally, where desired, insect cells may beutilized as host cells in the method of the present invention. See, e.g.Miller et al, Genetic Engineering, 8:277-298 (Plenum Press 1986) andreferences cited therein.

Another aspect of the present invention provides vectors for use in themethod of expression of these tendon/ligament-like tissue inducingproteins. Preferably the vectors contain the full novel DNA sequencesdescribed above which encode the novel factors of the invention.Additionally, the vectors contain appropriate expression controlsequences permitting expression of the protein sequences. Alternatively,vectors incorporating modified sequences as described above are alsoembodiments of the present invention. Additionally, the sequence of SEQID NO:1 or SEQ ID NO:3 or SEQ ID NO:25 could be manipulated to express amature protein by deleting propeptide sequences and replacing them withsequences encoding the complete propeptides of BMP proteins or membersof the TGF-β superfamily. Thus, the present invention includes chimericDNA molecules encoding a propeptide from a member of the TGF-βsuperfamily linked in correct reading frame to a DNA sequence encoding aprotein having the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4 orSEQ ID NO:26. The vectors may be employed in the method of transformingcell lines and contain selected regulatory sequences in operativeassociation with the DNA coding sequences of the invention which arecapable of directing the replication and expression thereof in selectedhost cells. Regulatory sequences for such vectors are known to thoseskilled in the art and may be selected depending upon the host cells.Such selection is routine and does not form part of the presentinvention.

A protein of the present invention, which induces tendon/ligament-liketissue or other tissue formation in circumstances where such tissue isnot normally formed, has application in the healing of tendon orligament tears, deformities and other tendon or ligament defects inhumans and other animals. Such a preparation employing atendon/ligament-like tissue inducing protein may have prophylactic usein preventing damage to tendon or ligament tissue, as well as use in theimproved fixation of tendon or ligament to bone or other tissues, and inrepairing defects to tendon or ligament tissue. De novotendon/ligament-like tissue formation induced by a composition of thepresent invention contributes to the repair of congenital, traumainduced, or other tendon or ligament defects of other origin, and isalso useful in cosmetic plastic surgery for attachment or repair oftendons or ligaments. The compositions of the invention may also beuseful in the treatment of tendinitis, carpal tunnel syndrome and othertendon or ligament defects. The compositions of the present inventioncan also be used in other indications wherein it is desirable to heal orregenerate tendon and/or ligament tissue. Such indications include,without limitation, regeneration or repair of injuries to theperiodontal ligament, such as occurs in tendonitis, and regeneration orrepair of the tendon-to-bone attachment. The compositions of the presentinvention may provide an environment to attract tendon- orligament-forming cells, stimulate growth of tendon- or ligament-formingcells or induce differentiation of progenitors of tendon- orligament-forming cells.

The BMP-12 related proteins may be recovered from the culture medium andpurified by isolating them from other proteinaceous materials from whichthey are co-produced and from other contaminants present. The proteinsof the present invention are capable of inducing the formation oftendon/ligament-like tissue. These proteins may be further characterizedby the ability to demonstrate tendon/ligament-like tissue formationactivity in the rat ectopic implant assay described below. It iscontemplated that these proteins may have ability to induce theformation of other types of tissue, such as ligaments, as well.

The tendon/ligament-like tissue inducing proteins provided herein alsoinclude factors encoded by the sequences similar to those of SEQ ID NO:1or SEQ ID NO:25, but into which modifications are naturally provided(e.g. allelic variations in the nucleotide sequence which may result inamino acid changes in the polypeptide) or deliberately engineered. Forexample, synthetic polypeptides may wholly or partially duplicatecontinuous sequences of the amino acid residues of SEQ ID NO:2. Thesesequences, by virtue of sharing primary, secondary, or tertiarystructural and conformational characteristics with tendon/ligament-liketissue growth factor polypeptides of SEQ ID NO:2 may possesstendon/ligament-like or other tissue growth factor biological propertiesin common therewith. Thus, they may be employed as biologically activesubstitutes for naturally-occurring tendon/ligament-like tissue inducingpolypeptides in therapeutic compositions and processes.

Other specific mutations of the sequences of tendon/ligament-like tissueinducing proteins described herein involve modifications ofglycosylation sites. These modifications may involve O-linked orN-linked glycosylation sites. For instance, the absence of glycosylationor only partial glycosylation results, from amino acid substitution ordeletion at asparagine-linked glycosylation recognition sites. Theasparagine-linked glycosylation recognition sites comprise tripeptidesequences which are specifically recognized by appropriate cellularglycosylation enzymes. These tripeptide sequences may beasparagine-X-threonine, asparagine-X-serine or asparagine-X-cysteine,where X is usually any amino acid except proline. A variety of aminoacid substitutions or deletions at one or both of the first or thirdamino acid positions of a glycosylation recognition site (and/or aminoacid deletion at the second position) results in non-glycosylation atthe modified tripeptide sequence. Additionally, bacterial expression ofprotein will also result in production of a non-glycosylated protein,even if the glycosylation sites are left unmodified.

The compositions of the present invention comprise a purified BMP-12related protein which may be produced by culturing a cell transformedwith the DNA sequence of SEQ ID NO:1 or SEQ ID NO:25 and recovering andpurifying protein having the amino acid sequence of SEQ ID NO:2 or SEQID NO:26 from the culture medium. The purified expressed protein issubstantially free from other proteinaceous materials with which it isco-produced, as well as from other contaminants. The recovered purifiedprotein is contemplated to exhibit tendon/ligament-like tissue formationactivity, and other tissue growth activity, such as ligamentregeneration. The proteins of the invention may be further characterizedby the ability to demonstrate tendon/ligament-like tissue formationactivity in the rat assay described below.

The compositions for inducing tendon/ligament-like tissue formation ofthe present invention may comprise an effective amount of atendon/ligament-like tissue inducing protein, wherein said proteincomprises the amino acid sequence of SEQ ID NO:2, preferably amino acids#-25, #1 or #3 to #103 or #104 of SEQ ID NO:2; or amino acids #1 or #19to #120 of SEQ ID NO:26; as well as mutants and/or variants of SEQ IDNO:2 or SEQ ID NO:26, which exhibit the ability to form tendon and/orligament like tissue.

Compositions of the present invention may further comprise additionalproteins, such as additional members of the TGF-β superfamily ofproteins, such as activins. Another aspect of the invention providespharmaceutical compositions containing a therapeutically effectiveamount of a tendon/ligament-inducing protein, such as BMP-12 or VL-1, ina pharmaceutically acceptable vehicle or carrier. These compositions maybe used to induce the formation of tendon/ligament-like tissue or othertissue. It is contemplated that such compositions may also be used fortendon and ligament repair, wound healing and other tissue repair, suchas skin repair. It is further contemplated that proteins of theinvention may increase neuronal survival and therefore be useful intransplantation and treatment of conditions exhibiting a decrease inneuronal survival. Compositions of the invention may further include atleast one other therapeutically useful agent, such as the BMP proteinsBMP-1, BMP-2, BMP-3, BMP4, BMP-5, BMP-6 and BMP-7, disclosed forinstance in U.S. Pat. Nos. 5,108,922; 5,013,649; 5,116,738; 5,106,748;5,187,076: and 5,141,905; BMP-8, disclosed in PCT publicationWO91/18098; BMP-9, disclosed in PCT publication WO93/00432; and BMP-10or BMP-11, disclosed in co-pending patent applications, Ser. Nos.08/061,695 and 08/061,464, filed on May 12, 1993. The disclosure of theabove documents are hereby incorporated by reference herein.

The compositions of the invention may comprise, in addition to atendon/ligament-inducing protein such as BMP-12 or VL-1 (BMP-13), othertherapeutically useful agents including MP52, epidermal growth factor(EGF), fibroblast growth factor (FGF), platelet derived growth factor(PDGF), transforming growth factors (TGF-α and TGF-β), and fibroblastgrowth factor-4 (FGF4), parathyroid hormone (PTH), leukemia inhibitoryfactor (LIF/HILDA/DIA), insulin-like growth factors (IGF-I and IGF-II).Portions of these agents may also be used in compositions of the presentinvention. For example, a composition comprising both BMP-2 and BMP-12implanted together gives rise to both bone and tendon/ligament-liketissue. Such a composition may be useful for treating defects of theembryonic joint where tendon, ligaments, and bone form simultaneously atcontiguous anatomical locations, and may be useful for regeneratingtissue at the site of tendon attachment to bone. It is contemplated thatthe compositions of the invention may also be used in wound healing,such as skin healing and related tissue repair. The types of woundsinclude, but are not limited to burns, incisions and ulcers. (See, e.g.PCT Publication WO84/01106 for discussion of wound healing and relatedtissue repair).

It is expected that the proteins of the invention may act in concertwith or perhaps synergistically with other related proteins and growthfactors. Further therapeutic methods and compositions of the inventiontherefore comprise a therapeutic amount of at least one protein of theinvention with a therapeutic amount of at least one of the BMP proteinsdescribed above. Such compositions may comprise separate molecules ofthe BMP proteins or heteromolecules comprised of different BMP moieties.For example, a method and composition of the invention may comprise adisulfide linked dimer comprising a BMP-12 related protein subunit and asubunit from one of the “BMP” proteins described above. Thus, thepresent invention includes compositions comprising a purified BMP-12related polypeptide which is a heterodimer wherein one subunit comprisesthe amino acid sequence from amino acid #1 to amino acid #104 of SEQ IDNO:2, and one subunit comprises an amino acid sequence for a bonemorphogenetic protein selected from the group consisting of BMP-1,BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, BMP-10 andBMP-11. A further embodiment may comprise a heterodimer of disulfidebonded tendon/ligament-like tissue inducing moieties such as BMP-12,VL-1 (BMP-13) or MP52. For example the heterodimer may comprise onesubunit comprising an amino acid sequence from #1 to # 104 of SEQ IDNO:2 and the other subunit may comprise an amino acid sequence from #1to #120 of SEQ ID NO:4 or #1 to #120 of SEQ ID NO:26. Further,compositions of the present invention may be combined with other agentsbeneficial to the treatment of the defect, wound, or tissue in question.

The preparation and formulation of such physiologically acceptableprotein compositions, having due regard to pH, isotonicity, stabilityand the like, is within the skill of the art. The therapeuticcompositions are also presently valuable for veterinary applications dueto the lack of species specificity in TGF-β proteins. Particularlydomestic animals and thoroughbred horses in addition to humans aredesired patients for such treatment with the compositions of the presentinvention.

The therapeutic method includes administering the composition topically,systemically, or locally as an injectable and/or implant or device. Whenadministered, the therapeutic composition for use in this invention is,of course, in a pyrogen-free, physiologically acceptable form. Further,the composition. may desirably be encapsulated or injected in a viscousform for delivery to the site of tissue damage. Topical administrationmay be suitable for wound healing and tissue repair. Therapeuticallyuseful agents other than the proteins which may also optionally beincluded in the composition as described above, may alternatively oradditionally, be administered simultaneously or sequentially with thecomposition in the methods of the invention. In addition, thecompositions of the present invention may be used in conjunction withpresently available treatments for tendon/ligament injuries, such assuture (e.g., vicryl sutures or surgical gut sutures, Ethicon Inc.,Somerville, N.J.) or tendon/ligament allograft or autograft, in order toenhance or accelerate the healing potential of the suture or graft. Forexample. the suture, allograft or autograft may be soaked in thecompositions of the present invention prior to implantation. It may alsobe possible to incorporate the protein or composition of the inventiononto suture materials, for example, by freeze-drying.

The compositions may include an appropriate matrix and/or sequesteringagent as a carrier. For instance, the matrix may support the compositionor provide a surface for tendon/ligament-like tissue formation and/orother tissue formation. The matrix may provide slow release of theprotein and/or the appropriate environment for presentation thereof. Thesequestering agent may be a substance which aids in ease ofadministration through injection or other means, or may slow themigration of protein from the site of application.

The choice of a carrier material is based on biocompatibility,biodegradability, mechanical properties, cosmetic appearance andinterface properties. The particular application of the compositionswill define the appropriate formulation. Potential matrices for thecompositions may be biodegradable and chemically defined. Furthermatrices are comprised of pure proteins or extracellular matrixcomponents. Other potential matrices are nonbiodegradable and chemicallydefined. Preferred matrices include collagen-based materials, includingsponges, such as Helistat® (Integra LifeSciences, Plainsboro, N.J.), orcollagen in an injectable form, as well as sequestering agents, whichmay be biodegradable, for example hyalouronic acid derived.Biodegradable materials, such as cellulose films, or surgical meshes,may also serve as matrices. Such materials could be sutured into aninjury site, or wrapped around the tendon/ligament.

Another preferred class of carrier are polymeric matrices, includingpolymers of poly(lactic acid), poly(glycolic acid) and copolymers oflactic acid and glycolic acid. These matrices may be in the form of asponge, or in the form of porous particles, and may also include asequestering agent. Suitable polymer matrices are described, forexample, in WO93/00050, the disclosure of which is incorporated hereinby reference.

Preferred families of sequestering agents include blood, fibrin clotand/or cellulosic materials such as alkylcelluloses (includinghydroxyalkylcelluloses), including methylcellulose, ethylcellulose,hydroxyethylcellulose. hydroxypropylcellulose,hydroxypropyl-methylcellulose, and carboxymethylcellulose. the mostpreferred being cationic salts of carboxymethylcellulose (CMC). Otherpreferred sequestering agents include hyaluronic acid, sodium alginate,poly(ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer andpoly(vinyl alcohol). The amount of sequestering agent useful herein is0.5-20 wt %, preferably 1-10 wt % based on total formulation weight;which represents the amount necessary to prevent desorbtion of theprotein from the polymer matrix and to provide appropriate handling ofthe composition, yet not so much that the progenitor cells are preventedfrom infiltrating the matrix, thereby providing the protein theopportunity to assist the activity of the progenitor cells.

Additional optional components useful in the practice of the subjectapplication include, e.g. cryogenic protectors such as mannitol,sucrose, lactose, glucose, or glycine (to protect the protein fromdegradation during lyophilization), antimicrobial preservatives such asmethyl and propyl parabens and benzyl alcohol; antioxidants such asEDTA, citrate and BHT (butylated hydroxytoluene); and surfactants suchas poly(sorbates) and poly(oxyethylenes); etc.

As described above, the compositions of the invention may be employed inmethods for treating a number of tendon defects, such as theregeneration of tendon/ligament-like tissue in areas of tendon orligament damage, to assist in repair of tears of tendon tissue,ligaments, and various other types of tissue defects or wounds. Thesemethods, according to the invention, entail administering to a patientneeding such tendon/ligament-like tissue or other tissue repair, acomposition comprising an effective amount of a tendon/ligament-liketissue inducing protein, such as described in SEQ ID NO:2, SEQ ID NO:4and/or SEQ ID NO:26. These methods may also entail the administration ofa tendon/ligament-like tissue inducing protein in conjunction with atleast one of the BMP proteins described above.

In another embodiment, the methods may entail administration of aheterodimeric protein in which one of the monomers is atendon/ligament-like tissue inducing polypeptide, such as BMP-12, VL-1(BMP-13) or MP52, and the second monomer is a member of the TGF-βsuperfamily of growth factors. In addition, these methods may alsoinclude the administration of a tendon/ligament-like tissue inducingprotein with other growth factors including EGF, FGF, TGF-α. TGF-β. andIGF.

Thus, a further aspect of the invention is a therapeutic method andcomposition for repairing tendon/ligament-like tissue, for repairingtendon or ligament as well as treating tendinitis and other conditionsrelated to tendon or ligament defects. Such compositions comprise atherapeutically effective amount of one or more tendon/ligament-liketissue inducing proteins, such as BMP-12, a BMP-12 related protein, orMP52, in admixture with a pharmaceutically acceptable vehicle, carrieror-matrix.

The dosage regimen will be determined by the attending physicianconsidering various factors which modify the action of the composition,e.g., amount of tendon or ligament tissue desired to be formed, the siteof tendon or ligament damage, the condition of the damaged tendon orligament, the size of a wound, type of damaged tissue, the patient'sage, sex, and diet, the severity of any infection, time ofadministration and other clinical factors. The dosage may vary with thetype of matrix used in the reconstitution and the types of additionalproteins in the composition. The addition of other known growth factors,such as IGF-I (insulin like growth factor I), to the final composition,may also affect the dosage.

Progress can be monitored by periodic assessment of tendon/ligament-liketissue formation, or tendon or ligament growth and/or repair. Theprogress can be monitored by methods known in the art, for example,X-rays, arthroscopy, histomorphometric determinations and tetracyclinelabeling.

The following examples illustrate practice of thee present invention inrecovering and characterizing human tendon/ligament-like tissue inducingprotein and employing them to recover the other tendon/ligament-liketissue inducing proteins, obtaining the human proteins, expressing theproteins via recombinant techniques, and demonstration of the ability ofthe compositions of the present invention to form tendon/ligament-liketissue in an in vivo model. Although the examples demonstrate theinvention with respect to BMP-12, with minor modifications within theskill of the art, the same results are believed to be attainable withMP52 and VL-1.

EXAMPLE 1

Isolation of DNA

DNA sequences encoding BMP-12 and BMP-12 related proteins may beisolated by various techniques known to those skilled in the art. Asdescribed below, oligonucleotide primers may be designed on the basis ofamino acid sequences present in other BMP proteins, Vg-1 relatedproteins and other proteins of the TGF-β superfamily. Regions containingamino acid sequences which are highly conserved within the BMP family ofproteins and within other members of the TGF-β superfamily of proteinscan be identified and consensus amino acid sequences of these highlyconserved regions can be constructed based on the similarity of thecorresponding regions of individual BMP/TGF-β/Vg-1 proteins. An exampleof such a consensus amino acid sequence is indicated below.

Consensus amino acid sequence (1):

Trp-Gln/Asn-Asp-Trp-Ile-Val/Ile-Ala (SEQ ID NO:16)

Where X/Y indicates that either amino acid residue may appear at thatposition.

The following oligonucleotide is designed on the basis of the aboveidentified consensus amino acid sequence (1):

#1: CGGATCCTGGVANGAYTGGATHRTNGC (SEQ ID NO:17)

This oligonucleotide sequence is synthesized on an automated DNAsynthesizer. The standard nucleotide symbols in the above identifiedoligonucleotide primer are as follows: A,adenosine; C,cytosine;G,guanine; T,thymine; N,adenosine or cytosine or guanine or thymine;R,adenosine or cytosine; Y,cytosine or thymine; H,adenosine or cytosineor thymine; V,adenosine or cytosine or guanine; D,adenosine or guanineor thymine.

The first seven nucleotides of oligonucleotide #1 (underlined) containthe recognition sequence for the restriction endonuclease BamHI in orderto facilitate the manipulation of a specifically amplified DNA sequenceencoding the BMP-12 protein and are thus not derived from the consensusamino acid sequence (1) presented above.

A second consensus amino acid sequence is derived from another highlyconserved region of BMP/TGF-β/Vg-1 proteins as described below:

His-Ala-Ile-Val/Leu-Gln-Thr (SEQ ID NO:18)

The following oligonucleotide is designed on the basis of the aboveidentified consensus amino acid sequence (2):

#2: TTTCTAGAARNGTYTGNACDATNGCRTG (SEQ ID NO: 19)

This oligonucleotide sequence is synthesized on an automated DNAsynthesizer. The same nucleotide symbols are used as described above.

The first seven nucleotides of oligonucleotide #1 (underlined) containthe recognition sequence for the restriction endonuclease XbaI in orderto facilitate the manipulation of a specifically amplified DNA sequenceencoding the BMP-12 protein and are thus not derived from the consensusamino acid sequence (2) presented above.

It is contemplated that the BMP-12 protein of the invention and otherBMP/TGF-β/Vg-1 related proteins may contain amino acid sequences similarto the consensus amino acid sequences described above and that thelocation of those sequences within a BMP-12 protein or other novelrelated proteins would correspond to the relative locations in theproteins from which they were derived. It is further contemplated thatthis positional information derived from the structure of otherBMP/TGF-β/Vg-1 proteins and the oligonucleotide sequences #1 and #2which have been derived from consensus amino acid sequences (1) and (2),respectively, could be utilized to specifically amplify DNA sequencesencoding the corresponding amino acids of a BMP-12 protein or otherBMP/TGF-β/Vg-1 related proteins.

Based on the knowledge of the gene structures of BMP/TGF-β/Vg-1 proteinsit is further contemplated that human genomic DNA can be used as atemplate to perform specific amplification reactions which would resultin the identification of BMP-12 BMP/TGF-β/Vg-1 (BMP-12 related protein)encoding sequences. Such specific amplification reactions of a humangenomic DNA template could be initiated with the use of oligonucleotideprimers #1 and #2 described earlier. Oligonucleotides #1 and #2identified above are utilized as primers to allow the specificamplification of a specific nucleotide sequence from human genomic DNA.The amplification reaction is performed as follows:

Human genomic DNA (source: peripheral blood lymphocytes), provided byKen Jacobs of Genetics Institute, is sheared by repeated passage througha 25 gauge needle, denatured at 100° C. for 5 minutes and then chilledon ice before adding to a reaction mixture containing 200 μM eachdeoxynucleotide triphosphates (dATP. dGTP, dCTP and dTTP), 10 mMTris-HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl₂. 0.001% gelatin, 1.25 units TaqDNA polymerase, 100 pM oligonucleotide #1 and 100 pM oligonucleotide #2.This reaction mixture is incubated at 94° C. for two minutes and thensubjected to thermal cycling in the following manner: 1 minute at 94°C., 1 minute at 40° C., 1 minute at 72° C for three cycles; then 1minute at 94° C., 1 minute at 55° C., 1 minute at 72° C. for thirtyseven cycles, followed by a 10 minute incubation at 72° C.

The DNA which is specifically amplified by this reaction is ethanolprecipitated, digested with the restriction endonucleases BamHI and XbaIand subjected to agarose gel electrophoresis. A region of the gel,corresponding to the predicted size of the BMP-12 or otherBMP/TGF-β/Vg-1 encoding DNA fragment, is excised and the specificallyamplified DNA fragments contained therein are electroeluted andsubcloned into the plasmid vector pGEM-3 between the XbaI and BamHIsites of the polylinker. DNA sequence analysis of one of the resultingBMP-12 related subclones indicates the specifically amplified DNAsequence product contained therein encodes a portion of the BMP-12protein of the invention.

The DNA sequence (SEQ ID NO:5) and derived amino acid sequence (SEQ IDNO:6) of this specifically amplified DNA fragment of BMP-12 are shown inthe SEQUENCE Listings.

Nucleotides #1-#26 of SEQ ID NO:5 comprise a portion of oligonucleotide#1 and nucleotides #103-#128 comprise a portion of the reversecompliment of oligonucleotide #2 utilized to perform the specificamplification reaction. Due to the function of oligonucleotides #1 and#2 in initiating the amplification reaction, they may not correspondexactly to the actual sequence encoding a BMP-12 protein and aretherefore not translated in the corresponding amino acid derivation (SEQID NO:6).

DNA sequence analysis of another subclone indicates that thespecifically amplified DNA product contained therein encodes a portionof another BMP/TGF-β/Vg-1 (BMP-12 related) protein of the inventionnamed VL-1.

The DNA sequence (SEQ ID NO:7) and derived amino acid sequence (SEQ IDNO:8) of this specifically amplified DNA fragment are shown in theSequence Listings.

Nucleotides #1-#26 of SEQ ID NO:7 comprise a portion of oligonucleotide#1 and nucleotides #103-#128 comprise a portion of the reversecompliment of oligonucleotide #2 utilized to perform the specificamplification reaction. Due to the function of oligonucleotides #1 and#2 in initiating the amplification reaction, they may not correspondexactly to the actual sequence encoding a VL-1 protein of the inventionand are therefore not translated in the corresponding amino acidderivation (SEQ ID NO:8).

The following oligonucleotide probe is designed on the basis of thespecifically amplified BMP-12 human DNA sequence set forth above (SEQ IDNO:5) and synthesized on an automated DNA synthesizer:

#3: CCACTGCGAGGGCCTTTGCGACTTCCCTTTGCGTTCGCAC (SEQ ID NO:20)

This oligonucleotide probe is radioactively labeled with ³²p andemployed to screen a human genomic library constructed in the vectorλFIX (Stratagene catalog #944201). 500,000 recombinants of the humangenomic library are plated at a density of approximately 10,000recombinants per plate on 50 plates. Duplicate nitrocellulose replicasof the recombinant bacteriophage plaques and hybridized tooligonucleotide probe #3 in standard hybridization buffer (SHB=5×SSC,0.1 % SDS, 5× Denhardt's, 100 μg/ml salmon sperm DNA) at 65° C.overnight. The following day the radioactively labelled oligonucleotidecontaining hybridization solution is removed an the filters are. washedwith 0.2×SSC, 0.% SDS at 65° C. A single positively hybridizingrecombinant is identified and plaque purified. This plaque purifiedrecombinant bacteriophage clone which hybridizes to the BMP-12oligonucleotide probe #3 is designated λHuG-48. A bacteriophage platestock is made and bacteriophage DNA is isolated from the λHuG-48 humangenomic clone. The bacteriophage λHuG-48 has been deposited with theAmerican Type Culture Collection, 12301 Parklawn Drive, Rockville, Md.“ATCC” under the accession #75625 on Dec. 7, 1993. This deposit meetsthe requirements of the Budapest Treaty of the International Recognitionof the Deposit of Microorganisms for the Purpose of Patent Procedure andRegulations thereunder. The oligonucleotide hybridizing region of thisrecombinant, λHuG-48, is localized to a 3.2 kb BamHI fragment. Thisfragment is subcloned into a plasmid vector (pGEM-3) and DNA sequenceanalysis is performed. This plasmid subclone is designated PCR1-1#2 andhas been deposited with the American Type Culture Collection, 12301Parklawn Drive, Rockville, Md. “ATCC” under the accession #69517 on Dec.7, 1993. This deposit meets the requirements of the Budapest Treaty ofthe International Recognition of the Deposit-of-Microorganisms, for thePurpose of Patent Procedure and Regulations thereunder. The partial DNAsequence (SEQ ID NO:1) and derived amino acid sequence (SEQ ID NO:2) ofthe 3.2 kb DNA insert of the plasmid subclone PCR1-1#2, derived fromclone λHuG-48, are shown in the Sequence Listings.

It should be noted that nucleotides #639-#714 of SEQ ID NO:1 correspondto nucleotides #27-#102 of the specifically amplified BMP-12 encodingDNA fragment set forth in SEQ ID NO:5 thus confirming that the humangenomic bacteriophage clone λHuG-48 and derivative subclone PCR1-1#2encode at least a portion of the BMP-12 protein of the invention. Thenucleotide sequence of a portion of the 3.2 kb BamHI insert of theplasmid PCR1-1#2 contains an open reading frame of at least 882 basepairs, as defined by nucleotides #1-#882 of SEQ ID NO:1. This open,reading frame encodes at least 294 amino acids of the human BMP-12protein of the invention. The encoded 294 amino acid human BMP-12protein includes the full mature human BMP-12 protein (amino acids#1-#104 of SEQ ID NO:2), as well as the C-terminal portion of thepropeptide region of the primary translation product (amino acid #-190to #-1 of SEQ ID NO:2).

Additional DNA sequence of the 3.2 kb BamHI insert of the plasmidP,CR1-1#2 set forth in SEQ ID NO:33 demonstrates the presence of an 1164bp open reading frame, as defined by nucleotides #138 through #1301 ofSEQ ID NO:33. [NOTE that all the sequence disclosed in SEQ ID NO:1 iscontained within SEQ ID NO:33]. As this sequence is derived from agenomic clone it is difficult to determine the boundary between the 5′extent of coding sequence and the 3′ limit of intervening sequence(intron/non-coding sequence).

Based on the knowledge of other BMP proteins and other proteins withinthe TGF-β family, it is predicted that the precursor polypeptide wouldbe cleaved at the multibasic sequence Arg-Arg-Gly-Arg in agreement witha proposed consensus proteolytic processing sequence of Arg-X-X-Arg.Cleavage of the BMP-12 precursor polypeptide is expected to generate a104 amino acid mature peptide beginning with the amino acid Ser atposition #1 of SEQ ID NO:2. The processing of BMP-12 into the matureform is expected to involve dimerization and removal of the N-terminalregion in a manner analogous to the processing of the related proteinTGF-β [Gentry et al., Molec & Cell. Biol., 8:4162 (1988); Derynck:et al.Nature, 316:701 (1985)].

It is contemplated therefore that the mature active species of BMP-12comprises a homodimer of two polypeptide subunits, each subunitcomprising amino acids #1 to #104 of SEQ ID NO:2 with a predictedmolecular weight of approximately 12,000 daltons. Further active speciesare contemplated comprising at least amino acids #3 to #103 of SEQ IDNO:2, thereby including the first and last conserved cysteine residue.As with other members of the TGF-β/BMP family of proteins, thecarboxy-terminal portion of the BMP-12 protein exhibits greater sequenceconservation than the more amino-terminal portion. The percent aminoacid identity of the human BMP-12 protein in the cysteine-richC-terminal domain (amino acids #3-#104) to the corresponding region ofhuman BMP proteins and other proteins within the TGF-β family is asfollows: BMP-2, 55%; BMP-3, 43%; BMP-4, 53%; BMP-5, 49%; BMP-6, 49%;BMP-7, 50%; BMP-8, 57%; BMP-9, 48%; BMP-10, 57%; activin WC (BMP-11),38%; Vg1, 46%; GDF-1, 47%; TGF-β1, 36%; TGF-β2, 36%; TGF-β3, 39%;inhibin β(B), 36%; inhibin β(A), 41%.

The human BMP-12 DNA sequence (SEQ ID NO:1), or a portion thereof, canbe used as a probe to identify a human cell line or tissue whichsynthesizes BMP-12 mRNA. Briefly described, RNA is extracted from aselected cell or tissue source and either electrophoresed on aformaldehyde agarose gel and transferred to nitrocellulose, or reactedwith formaldehyde and spotted on nitrocellulose directly. Thenitrocellulose is then hybridized to a probe derived from the codingsequence of human BMP-12.

Alternatively, the human BMP-12 sequence is used to designoligonucleotide primers which will specifically amplify a portion of theBMP-12 encoding sequence located in the region between the primersutilized to perform the specific amplification reaction. It iscontemplated that these human BMP-12 derived primers would allow one tospecifically amplify corresponding BMP-12 encoding sequences from mRNA,cDNA or genomic DNA templates. Once a positive source has beenidentified by one of the above described methods, mRNA is selected byoligo (dT) cellulose chromatography and cDNA is synthesized and clonedin λgt10 or other λ bacteriophage vectors known to those skilled in theart, for example, λZAP by established techniques (Toole et al., supra).It is also possible to perform the oligonucleotide primer directedamplification reaction, described above, directly on a pre-establishedhuman cDNA or genomic library which has been cloned into a λbacteriophage vector. In such cases, a library which yields aspecifically amplified DNA product encoding a portion of the humanBMP-12 protein could be screened directly, utilizing the fragment ofamplified BMP-12 encoding DNA as a probe.

Oligonucleotide primers designed on the basis of the DNA sequence of thehuman BMP-12 genomic clone λHuG-48 are predicted to allow the specificamplification of human BMP-12 encoding DNA sequences frompre-established human cDNA libraries which are commercially available(ie. Stratagene, La Jolla, Calif. or Clontech Laboratories, Inc., PaloAlto, Calif.). The following oligonucleotide primer is designed on thebasis of nucleotides #571 to #590 of the DNA sequence set forth in SEQID NO:1 and synthesized on an automated DNA synthesizer: #4:TGCGGATCCAGCCGCTGCAGCCGCAAGCC (SEQ ID NO:21)

The first nine nucleotides of primer #4 (underlined) comprise therecognition sequence for the restriction endonuclease BamHI which can beused to facilitate the manipulation of a specifically amplified DNAsequence encoding the human BMP-12 protein of the invention and are thusnot derived from the DNA sequence presented in SEQ ID NO:1.

The following oligonucleotide primer is designed on the basis ofnucleotides #866-#885 of the DNA sequence set forth in SEQ ID NO:1 andsynthesized on an automated DNA synthesizer: #5GACTCTAGACTACCTGCAGCCGCAGGCCT (SEQ ID NO:22)

The first nine nucleotides of primer #5 (underlined) comprise therecognition sequence for the restriction endonuclease Xbal which can beused to facilitate the manipulation of a specifically amplified DNAsequence encoding the human BMP-12 protein of the invention and are thusnot derived from the DNA sequence presented in SEQ ID NO:1.

The standard nucleotide symbols in the above identified primers are asfollows: A, adenine; C, cytosine; G, guanine; T, thymine.

Primers #4 and #5 identified above are utilized as primers to allow theamplification of a specific BMP-12 encoding nucleotide sequence frompre-established cDNA libraries which may include the following: humanfetal brain cDNA/λZAPII (Stratagene catalog #936206), humanliver/λUNI-ZAP XR (Stratagene Catalog #937200), human lung/λUNI-ZAP XR(Stratagene catalog #937206), and human fetal spleen/UNI-ZAP XR(Stratagene catalog #937205).

Approximately 1×10⁸ pfu (plaque forming units) of λbacteriophagelibraries containing human cDNA inserts such as those detailed above aredenatured at 95° C. for five minutes prior to addition to a reactionmixture containing 200 μM each deoxynucleotide triphosphates (dATP,dGTP, dCTP and dTTP) 10 mM Tris-HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl₂,0.001% gelatin, 1.25 units Taq DNA polymerase, 100 pM oligonucleotideprimer #4 and 100 pM oligonucleotide primer #5. The reaction mixture isthen subjected to thermal cycling in the following manner: 1 minute at94° C., 1 minute at 50° C., 1 minute at 72° C. for thirty-nine cyclesfollowed by 10 minutes at 72° C.

The DNA which is specifically amplified by this reaction would beexpected to generate a BMP-12 encoding product of approximately 333 basepairs, the internal 315 bp of which correspond to nucleotides #571 to#885 of SEQ ID NO:1 and also including 9 bp at each end of the BMP-12specific fragment which correspond to the restriction sites defined bynucleotides #1-#9 of primers #4 and #5. The resulting. 333 bp DNAproduct is digested with the restriction endonucleases BamHI and XbaI,phenol extracted, chloroform extracted and ethanol precipitated.

Alternatively, to ethanol precipitation, buffer exchange and removal ofsmall fragments of DNA resulting from the BamHI/XbaI restriction digestis accomplished by dilution of the digested DNA product in 10 mMTris-HCl pH 8.0, 1 mM EDTA followed by centrifugation through aCentricon™ 30 microconcentrator (W.R. Grace & Co., Beverly, Mass.;Product #4209). The resulting BamHI/XbaI digested amplified DNA productis subcloned into a plasmid vector (ie. pBluescript, pGEM-3 etc.)between the BamHI and XbaI sites of the polylinker region. DNA sequenceanalysis of the resulting subclones would be required to confirm theintegrity of the BMP-12 encoding insert. Once a positive cDNA source hasbeen identified in this manner, the corresponding cDNA library fromwhich a 333 bp BMP-12 specific sequence was amplified could be screeneddirectly with the 333 bp insert or other BMP-12 specific probes in orderto identify and isolate cDNA clones encoding the full-length BMP-12protein-of the-invention.

Additional methods known to those skilled in the art may be used toisolate other full-length cDNAs encoding human BMP-12 related proteins,or full length cDNA clones encoding BMP-12 related proteins of theinvention from species other than humans, particularly other mammalianspecies.

The following examples demonstrate the use of the human BMP-12 sequenceto isolate homologues from BMP-12 related proteins in a murine genomicDNA library.

The DNA sequence which encodes the human BMP-12 protein of the inventionis predicted to be significantly homologous to BMP-12 and BMP-12 relatedsequences from species other than humans that it could be utilized tospecifically amplify DNA sequences from those other species which wouldencode the corresponding BMP-12 related proteins. Specifically, thefollowing oligonucleotides are designed on the basis of the human BMP-12sequence (SEQ ID NO:1) and are synthesized on an automated DNAsynthesizer: #6: GCGGATCCAAGGAGCTCGGCTGGGACGA (SEQ ID NO:23) #7:GGAATTCCCCACCACCATGTCCTCGTAT (SEQ ID NO:24)

The first eight nucleotides of oligonucleotide primers #6 and #7(underlined) comprise the recognition sequence for the restrictionendonucleases BamHI and EcoRI, respectively. These sequences areutilized to facilitate the manipulation of a specifically amplified DNAsequence encoding a BMP-12 or BMP-12 related protein from a speciesother than human and are thus not derived from the DNA sequencepresented in SEQ ID NO:1. Oligonucleotide primer #6 is designed on thebasis of nucleotides #607-#626 of SEQ ID NO:1. Oligonucleotide primer #7is designed on the basis of the reverse compliment of nucleotides#846-#865 of the DNA sequence set forth in SEQ ID NO:1.

Oligonucleotide primers #6 and #7 identified above are utilized asprimers to allow the amplification of specific BMP-12 related sequencesfrom genomic DNA derived from species other than humans. Theamplification reaction is performed as follows:

Murine genomic DNA (source: strain Balb c) is sheared by repeatedpassage through a 25 gauge needle, denatured at 100° C. for five minutesand then chilled on ice before adding to a reaction -mixture containing200 μM each deoxynucleotide triphosphates (dATP, DGTP, dCTP and dTTP) 10nM Tris-HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl₂, 0.001% gelatin, 1.25 unitsTaq DNA polymerase, 100 pM oligonucleotide primer #6 and 100 pM-oligonucleotide primer #7. The reaction mixture is then subjected tothermal cycling in the following manner: 1 minute at 95° C., 1 minute at55° C., 1 minute at 72° C. for forty cycles followed by 10 minutes at72° C.

The DNA which is specifically amplified by this reaction is ethanolprecipitated, digested with the restriction endonucleases BamHI andEcoRI and subjected to agarose gel electrophoresis. A region of the gel,corresponding to the predicted size of the murine BMP-12 or BMP-12related encoding DNA fragment, is excised and the specifically amplifiedDNA fragments contained therein are extracted (by electroelution or byother methods known to those skilled in the art) and subcloned in to aplasmid vector, such as pGEM-3 or pBluescript between the BaniHI andEcoRI sites of the polylinker. DNA sequence analysis of one of theresulting subclones named mV1, indicates that the specifically amplifiedDNA sequence contained therein encodes a portion of a protein whichappears to be the murine homolog to either the BMP-12 or VL-1 sequenceof the invention. The DNA sequence (SEQ ID NO:10) and derived amino acidsequence (SEQ ID NO:1 1) of this specifically amplified murine DNAfragment are shown in the sequence listings.

Nucleotides #1-#26 of SEQ ID NO:10 comprise a portion of oligonucleotide#6 and nucleotides #246-#272 comprise a portion of the reversecompliment of oligonucleotide #7 utilized to perform the specificamplification reaction. Nucleotide #27 of SEQ ID NO:10 appears to be thelast nucleotide of a codon triplet, and nucleotides #244-#245 of SEQ IDNO:10 appear to be the first two nucleotides of a codon triplet.Therefore, nucleotides #28 to #243 of SEQ ID NO:10 correspond to apartial coding sequence of mV1. Due to the function of oligonucleotides#6 and #7 in initiating the amplification reaction, they may notcorrespond exactly to the actual sequence encoding the murine homolog tothe human BMP-12 or VL-1 protein of the invention and are therefore nottranslated in the corresponding amino acid sequence derivation (SEQ IDNO:11).

Oligonucleotide probes designed on the basis of the specificallyamplified murine BMP-12 or VL-1 DNA sequence set forth-in SEQ ID NO:10can be utilized by those skilled in the art to identify full-lengthmurine BMP-12 or VL1 encoding clones (either cDNA or genomic).

DNA sequence analysis of another of the resulting subclones named mV2,indicates that the specifically amplified DNA sequence contained thereinencodes a portion of a murine BMP-12 related sequence of the invention.The DNA sequence (SEQ ID NO:12) and derived amino acid sequence (SEQ IDNO:13) of this specifically amplified murine DNA fragment are shown inthe sequence listings.

Nucleotides #1-#26 of SEQ ID NO:12 comprise a portion of oligonucleotide#6 and nucleotides #246-#272 comprise a portion of the reversecompliment of oligonucleotides #7 utilized to perform the specificamplification reaction. Nucleotide #27 of SEQ ID NO:12 appears to be thelast nucleotide of a codon triplet, and nucleotides #244-#245 of SEQ IDNO:12 appear to be the first two nucleotides of a codon triplet.Therefore, nucleotides #28 to #243 of SEQ ID NO:12 correspond to apartial coding sequence of mV2. Due to the function of oligonucleotides#6 and #7 in initiating the amplification reaction, they may notcorrespond exactly to the actual sequence encoding the murine BMP-12related protein of the invention and are therefore not translated in thecorresponding amino acid sequence derivation. (SEQ ID NO:13).

Oligonucleotide probes designed on the basis of the specificallyamplified murine BMP-12 related DNA sequence set forth in SEQ ID NO:12can be utilized by those skilled in the art to identify full-lengthmurine BMP-12 related encoding clones (either cDNA or genomic).

DNA sequence analysis of another of the resulting subclones named mV9,indicates that the specifically amplified DNA sequence contained thereinencodes a portion of a murine BMP-12 related sequence of the invention.This sequence appears to be the murine homolog to the human MP52 DNAsequence described at SEQ ID NO:3. The DNA sequence (SEQ ID NO:14) andderived amino acid sequence (SEQ ID NO:15) of this specificallyamplified murine DNA fragment are shown in the sequence listings.

Nucleotides #1-#26 of SEQ ID NO:14 comprise a portion of oligonucleotide#6 and nucleotides #246-#272 comprise a portion of the reversecompliment of oligonucleotide #7 utilized to perform the specificamplification reaction. Nucleotide #27 of SEQ ID NO:14 appears to be thelast nucleotide of a codon triplet, and nucleotides #244-#245 of SEQ IDNO:14 appear to be the first two nucleotides of a codon triplet.Therefore, nucleotides #28 to #243 of SEQ ID NO:14 correspond to apartial coding sequence of mV9. Due to the function of oligonucleotides#6 and #7 in initiating the amplification reaction, they may notcorrespond exactly to the actual sequence encoding the murine BMP-12related protein of the invention and are therefore not translated in thecorresponding amino acid sequence derivation (SEQ ID NO:15).

Oligonucleotide probes designed on the basis of the specificallyamplified murine BMP-12 related DNA sequence set forth in SEQ ID NO:14can be utilized by those skilled in the art to identify full-lengthmurine BMP-12 related encoding clones (either cDNA or genomic).

Alternatively, oligonucleotide primers #6 and #7 identified above areutilized as primers to allow the specific amplification of a 275 basepair DNA probe, the internal 259 bp of which correspond to nucleotides#607 to #865 of SEQ ID NO:1 from the BMP-12 encoding plasmid subclonePCR1-1#2. This 275 bp DNA probe was radioactively labelled with ³²p andemployed to screen a murine genomic library constructed in the vector λFIX II (Stratagene catalog #946306). 1 million recombinants of themurine genomic library are plated at a density of approximately 20,000recombinants per plate on 50 plates. Duplicate nitrocellulose replicasof the recombinant bacteriophage plaques are hybridized, under reducedstringency conditions, to the specifically amplified 333 bp probe instandard hybridization buffer (SHB=5×SSC, 0.1% SDS, 5× Denhardt's, 100μg/ml salmon sperm DNA) at 60° C. overnight. The following day theradioactively labelled oligonucleotide containing hybridization solutionis removed an the filters are washed, under reduced stringencyconditions, with 2×SSC, 0.1% SDS at 60° C. Multiple positivelyhybridizing recombinants are identified and plaque purified. Fragmentsof the positively hybridizing murine genomic recombinant clones aresubcloned into standard plasmid vectors (i.e. pGEM-3) and subjected toDNA sequence analysis.

DNA sequence analysis of one of these subclones named MVR3 indicatesthat it encodes a portion of the mouse gene corresponding to the PCRproduct mV1 (murine homolog of the human. BMP-12 sequence set forth inSEQ ID NO:1) described above. The partial DNA sequence of this subcloneand corresponding. amino acid translation are set forth in SEQ ID NO: 29and SEQ ID NO:30 respectively.

DNA sequence analysis of another one of these subclones named MVR32indicates that it encodes a portion of the mouse gene corresponding tothe PCR product mV2 (murine homolog of the human VL-1 sequence set forthin SEQ ID NO:7) described above. The partial DNA sequence of thissubclone and corresponding amino acid translation are set forth in SEQID NO: 31 and SEQ ID NO:32 respectively.

DNA sequence analysis of another of these subclones named MVR23indicates that it encodes a portion of the mouse gene corresponding tothe PCR product mV9 (murine homolog of the MP-52 sequence set forth inSEQ ID NO:3) described above.

In a similar manner to that which is described above for identifying andisolating human genomic clones encoding the BMP-12 protein of theinvention, oligonucleotide probe(s) corresponding to the VL-1 encodingsequence set forth in SEQ ID NO:7 can be designed and utilized toidentify human genomic or cDNA sequences encoding the VL-1 (BMP-13)protein. These oligonucleotides would be designed to regions specificfor VL-1 encoding sequences and would therefore be likely to be derivedfrom regions of the lowest degree of nucleotide sequence identitybetween the specifically amplified VL-1 encoding sequence (SEQ ID NO:7)and the specifically amplified BMP-12 encoding sequence (SEQ ID NO:5).

Alternatively, oligonucleotide primers #4 and #5 identified above areutilized as primers to allow the specific amplification of a 333 basepair DNA probe, the internal 315 bp of which correspond to nucleotides#571 to #885 of SEQ ID NO:1. from the BMP-12 encoding plasmid subclonePCR1-1#2. This 333 bp DNA probe was radioactively labelled with ³²p andemployed to screen a human genomic library constructed in the vectorλDASH II (Stratagene catalog #945203). 1 million recombinants of thehuman genomic library are plated at a density of approximately 20,000recombinants per plate on 50 plates. Duplicate nitrocellulose replicasof the recombinant bacteriophage plaques are hybridized, under reducedstringency conditions, to the specifically amplified 333 bp probe instandard hybridization buffer (SHB=5×SSC, 0.1 % SDS, 5× Denhardt's, 100μg/ml salmon sperm DNA) at 60° C. overnight. The following day theradioactively labelled oligonucleotide containing hybridization solutionis removed an the filters are washed, under reduced stringencyconditions, with 2×SSC, 0. 1% SDS at 60° C. Multiple (approximately 15)positively hybridizing recombinants are identified and plaque purified.

In order to distinguish positively hybridizing recombinants encoding theVL-1 protein of the invention from BMP-12 and other BMP-12-relatedencoding recombinants which would be predicted to hybridize positivelyto the 333 bp DNA probe generated from the BMP-12 encoding plasmidPCR1-1#2 utilized in this screening procedure, the followingoligonucleotide probe, based on the VL-1 sequence set forth in SEQ IDNO:7, is designed and synthesized on an automated DNA synthesizer:

#8: TGTATGCGACTTCCCGC [SEQUENCE ID NO: 35]

An oligonucleotide corresponding to nucleotides #60 to #76 of SEQ IDNO:7 which contains 5 nucleotide differences to the corresponding regionof the BMP-12 encoding sequence set forth in SEQ ID NO:1 (nucleotides#672 to #689) One of the recombinant bacteriophage clones whichhybridizes to the VL-1 oligonucleotide probe #8 is designated λJLDc31.This recombinant bacteriophage clone is plaque purified, a bacteriophageplate stock is made and bacteriophage DNA is isolated from the λJLDc31human genomic clone. The bacteriophage λJLDc3l has been deposited withthe American Type Culture Collection, 12301 Parklawn Drive, Rockville,Md. “ATCC” under the accession #75922 on Oct. 20, 1994. This depositmeets the requirements of the Budapest Treaty of the InternationalRecognition of the Deposit of Microorganisms for the Purpose of PatentProcedure and Regulations thereunder. The oligonucleotide hybridizingregion of this recombinant, λJLDc31, is localized to a 2.5 kb Eco RIfragment. This fragment is subcloned into a plasmid vector (pGEM-3) andDNA sequence analysis is performed. This plasmid subclone is designatedpGEMJLDc31/2.5 and has been deposited with the American Type CultureCollection, 12301 Parklawn Drive, Rockville, Md. “ATCC” under theaccession # 69710 on Oct. 20, 1994. This deposit meets the requirementsof the Budapest Treaty of the International Recognition of the Depositof Microorganisms for the Purpose of Patent Procedure and Regulationsthereunder.

The partial DNA sequence (SEQ ID NO:25) and:derived amino acid sequence(SEQ ID NO:26) of a portion of the 2.5 kb DNA insert of the plasmidsubclone pGEMJLDc31/2.5, derived from clone λJLDc31, are shown in theSequence Listings

The DNA sequence of a portion of the 2.5 kb EcoRI insert of the plasmidpGEMJLDc31/2.5 is set forth in SEQ ID NO:25. contains an 912 bp openreading frame, as defined by nucleotides #52 through #963 of SEQ IDNO:25. As this sequence is derived from a genomic clone it is difficultto determine the boundary between the 5′ extent of coding sequence andthe 3′ limit of intervening sequence (intron/non-coding sequence). Theentire open reading frame (nucleotides #52 through #963 of SEQ ID NO:25)encodes a portion of the VL1 protein of the invention of up to 304 aminoacids.

Based on the knowledge of other BMP proteins and other proteins withinthe TGF-β family, it is predicted that the precursor polypeptide wouldbe cleaved at the multibasic sequence Arg-Arg-Arg-Arg in agreement witha proposed consensus proteolytic processing sequence of Arg-X-X-Arg.Cleavage of the VL-1 precursor polypeptide is expected to generate a 120amino acid mature peptide beginning with, the amino acid Thr at position#1 of SEQ ID NO:26. The processing of VL-1 into the mature form isexpected to involve dimerization and removal of the N-terminal region ina manner analogous to the processing of the related protein TGF-β[Gentry et al., Molec & i Cell. Biol., 8:4162 (1988); Derynck et al.Nature, 316:701 (1985)].

It is contemplated therefore that the mature active species of VL-1comprises a homodimer of two polypeptide subunits, each subunitcomprising amino acids #1 to #120 of SEQ ID NO:26 with a predictedmolecular weight of approximately 12,000 daltons. Further active speciesare contemplated comprising at least amino acids #19 to # 119 or #120 ofSEQ ID NO:26, thereby including the first and last conserved cysteineresidue.

Using such a method, a clone encoding the mature human VL-1 (BMP-13) wasobtained. The nucleotide sequence and corresponding amino acid sequenceencoded by this clone are listed in the Sequence Listings at SEQ ID NO:25 and 26, respectively.

EXAMPLE 2

Expression of BMP-12

In order to produce human BMP-12 proteins, the DNA encoding it istransferred into an appropriate expression vector and introduced intomammalian cells or other preferred eukaryotic or prokaryotic hosts byconventional genetic engineering techniques.

In order to produce the human BMP-12 protein in bacterial cells, thefollowing procedure is employed.

Expression of BMP-12 in E. coli

An expression plasmid pALV1-78 1, for production of BMP-12 in E. coliwas constructed which contains the following principal features.Nucleotides 1-2060 contain DNA sequences originating from the plasmidpUC-18 [Norrander et al., Gene 26:101-106 (1983)] including sequencescontaining the gene for β-lactamase which confers resistance to theantibiotic ampicillin in host E. coli strains, and a colE1-derivedorigin of replication. Nucleotides 2061-2221 contain DNA sequences forthe major leftward promotor (pL) of bacteriophage λ [Sanger et al., J.Mol. Biol. 162:729-773 (1982)], including three operator sequences0_(L)1, 0_(L)2 and 0_(L)3. The operators are the binding sites for λcIrepressor protein, intracellular levels of which control the amount oftranscription initiation from pL. Nucleotides 2222-2723 contain a strongribosome binding sequence included on a sequence derived fromnucleotides 35566 to 35472 and 38137 to 38361 from bacteriophage lambdaas described in Sanger et al., J. Mol. Biol. 162:729-773 (1982).Nucleotides 2724-3041 contain a DNA sequence encoding mature BMP-12protein with all 3 untranslated sequence removed. The BMP-12 DNAsequences introduced into the pALV1-781 expression vector were modifiedat the 5′ end to raise the A+T content without altering the codingcapacity. These changes were made to increase the efficiency oftranslation initiated on the BMP-12 mRNA in E. coli. Nucleotides3042-3058 provide a “Linker” DNA sequence containing restrictionendonuclease sites. Nucleotides 3059-3127 provide a transcriptiontermination sequence based on that of the E. coli asp A gene [Takagi etal., Nucl. Acids Res. 13:2063-2074 (1985)]. Nucleotides 3128-3532 areDNA sequences derived from pUC-18.

Plasmid pALV1-781 was transformed into the E. coli host strain GI724 (F,lacI^(q), lacp^(L8), ampC::λcl⁺) by the procedure of Dagert and Ehrlich,Gene 6:23 (1979). GI724 (ATCC accession No. 55151) contains a copy ofthe wild-type λcI repressor gene stably integrated into the chromosomeat the ampC locus, where it has been placed under the transcriptionalcontrol of Salmonella typhimurium trp promotor/operator sequences. InGI724, λCI protein is made only during growth in tryptophan-free media,such as minimal media or a minimal medium supplemented with casaminoacids such as IMC, described above. Addition of tryptophan to a cultureof GI724 will repress the trp promoter and turn off synthesis of λcI,gradually causing the induction of transcription from pL promoters ifthey are present in the cell.

Transformants were selected on 1.5% w/v agar plates containing IMCmedium, which is composed of M9 medium [Miller, “Experiments inMolecular Genetics,” Cold Spring Harbor Laboratory, New York (1972)]containing 1 mM MgSO₄ and supplemented with 0.5% w/v glucose, 0.2% w/vcasamino acids and 100 μg/ml ampicillin. GI724 transformed withpALV1-781 was grown at 37° C. to an A₅₅₀ of 0.5 in IMC medium containing100 μg/ml ampicillin. Tryptophan was then added to a final concentrationof 100 μg/ml and the culture incubated for a further 4 hours. Duringthis time BMP-12 protein accumulates within the “inclusion body”fraction.

Preparation of Protein Monomer

18 g of frozen cells were weighed out and resuspended in 60 ml of 100 mMTris, 10 mM EDTA, 1 mM phenylmethylsulfonyl fluoride [PMSF], pH 8.3.Cells were lysed by 3 passes through a Microfluidizer™ [model #MCF 100T]. The inclusion body pellet was obtained by centrifugation at 15,000 gat 4° C. for 20 minutes. The supernatant was decanted, and the pelletwas washed with 100 ml of 100 mM Tris, 1.0 M NaCl, 10 mM EDTA, 1 mMPMSF, pH 8.3. The suspension was centrifuged again at 15,000 g at 4° C.for 10 minutes, and the supernatant decanted. The pellet was then washedwith 100 ml of 100 mM Tris, 10 mM EDTA. 1% Triton X-100, 1 mM PMSF, pH8.3. The suspension was centrifuged again at 15,000 g at 4° C. for 10minutes, and the supernatant decanted. The pellet was resuspended with50 ml of 20 mM Tris, 1 mM EDTA, 1 mM PMSF, pH 8.3, containing 1% DTT ina glass tissue homogenizer. Monomeric BMP-12 was then solubilized byacidification to pH 2.5 with glacial acetic acid. The soluble fractionwas isolated by centrifugation at 15,000 g for 20 minutes at 4° C.

The supernatant from this centrifugation was collected andchromatographed over a Sephacryl S-10™ size exclusion column (83 cm×2.6cm; ≈440 ml bed) in 20 ml increments. The Sephacryl S-100™ column wasrun with a mobile phase of 1% acetic acid at a flow rate of 1.4 ml/min.Fractions corresponding to BMP-12 monomer were detected by absorbance at280 nm, and using a computer calculated extinction coefficient of18200M⁻¹cm⁻¹ and molecular weight (11667 daltons). This size exclusioncolumn pooled material was used as starting material for refoldingreactions.

As an alternative to the above, 1.0 g of cells stored at −80° C. aremeasured. Solution (3.4 ml 100 mM TRIS:, 10 mM EDTA, pH 8.5) is added.The solution is vortexed until cells are well suspended. 40 μl 100 mMPMSF in isopropanol is added. The cells are lysed at 1000 psi in aFrench pressure cell. The inclusion bodies are centrifuged at 4° C. for20 minutes in an Eppendorf microfuge to form pellets. The supernatantsare decanted. To one pellet (out of 4 total) 1.0 ml degassed 8.0 Mguanidine hydrochloride, 0.5 M TRIS, 5 MM EDTA pH 8.5, containing 250 mMDTT is added. The pellet is dissolved and argon is blown over the liquidfor 30 seconds. Next the solution is incubated at 37° C. for one hour.Insoluble material is pelleted for 2-3 minutes in an Eppendorf microfugeat 23° C. 0.5-1.0 ml of supernatant is injected onto a Supelco 2 cmguard cartridge (LC-304), and eluted with an acetonitrile gradient in0.1% TFA from 1-70% over 35 minutes. BMP-12 elutes between 29 and 31minutes. Fractions are pooled and the protein concentration determinedby adsorbance at 280 nanometers versus 0.1% TFA, using the theoreticalextinction coefficient based upon the amino acid content. As a secondalternate method to the above, frozen cell pellets obtained from the E.coli transformants as described above are thawed in 30 ml ofTE8.3(100:10) buffer (100 mM Tris-HCl pH 8.3, 10 mM Na₂EDTA, 1 mM PMSF).Cells are lysed by three passes through a Microfluidizer™ [model #MCF100 T]. The initial inclusion body material pellet is dissolved in 8 Mguanidine-HCl, TE8.5(100:10) buffer (100 mM Tris-HCl pH 8.5, 10 mMNa₂EDTA which contained 100 mM DTT, and incubated at 37° C. for 1 hour.This-.material is centrifuged at 12,000×g for 15 minutes at roomtemperature.

Refolding of BMP-12 Protein Using CHAPS System

A sufficient volume of the BMP-12 pool is lyophilized to give 10 μg ofprotein. 5 μl of glass distilled water is added to redissolve theresidue, then 100 μl of refold mix (50 mM Tris, 1.0 M NaCl, 2%3-(3-chlolamido-propyl)dimethylammonio-1-propane-sulfate (CHAPS), 5 mMEDTA, 2 mM glutathione (reduced) 1 mM glutathione (oxidized); at pH ofapproximately 8.5). The solution is gently mixed and stored at 23° C.for 1-4 days. Dimer formation is assessed by running an aliquot on aNovex 16% tricine, gel at 125 volts for 2.5 hours, followed by CoomassieBlue staining and destaining.

BMP-12 dimer was purified using a C4 analytical RP-HPLC (reversedphase-high performance liquid chromatography) column (Vydac 214TP54)which was equilibrated to 1% B buffer (diluted into A buffer) and wasrun over 35 minutes, during which the protein elutes, using thefollowing gradient (A buffer=0.1% trifluoroacetic acid, B buffer=95%acetonitrile, 0.1% trifluoroacetic acid [TFA]), with a flow rate of 1ml/min:

1-5 minutes 20% B buffer

5-10 minutes 20-30% B buffer

10-30 minutes 30-50% B buffer

30-35 minutes 50-100% B buffer

Protein was monitored by absorbance at 280 nm. Peak BMP-12 fractions(eluting between 29 and 31 minutes) were pooled. Purity was assessed bySDS-PAGE. The concentration was determined by absorbance at 280 nm, andusing the computer calculated extinction coefficient and molecularweight as indicated above.

Expression of BMP-12 in Mammalian Cells:

Another contemplated preferred expression system for biologically activerecombinant human BMP-12 is stably transformed mammalian cells.

One skilled in the art can construct mammalian expression vectors byemploying the sequence of SEQ ID NO:1, or other DNA sequences encodingBMP-12 proteins or other modified sequences and known vectors, such aspCD [Okayama et al., Mol. Cell Biol., 2:161-170 (1982)], pJL3, pJL4[Gough et al., EMBO J., 4:645 -653 (1985)] and pMT2 CXM.

The mammalian expression vector pMT2 CXM is a derivative of p91023(b)(Wong et al., Science 228:810-815, 1985) differing from the latter inthat it contains the ampicillin resistance gene in place of thetetracycline resistance gene and further contains a XhoI site forinsertion of cDNA clones. The functional elements of pMT2 CXM have beendescribed (Kaufman, R. J., 1985, Proc. Natl. Acad. Sci. USA 82:689-693)and include the adenovirus VA genes, the SV40 origin of replicationincluding the 72 bp enhancer, the adenovirus major late promoterincluding a 5′ splice site and the majority of the adenovirus tripartiteleader sequence present on adenovirus late mRNAs, a 3′ splice acceptorsite, a DHFR insert, the SV40 early polyadenylation site (SV40), andpBP322 sequences needed for propagation in E. coli.

Plasmid pMT2 CXM is obtained by EcoRI digestion of pMT2-VWF, which hasbeen deposited with the American Type Culture Collection (ATCC),Rockville, Md. (USA) under accession number ATCC 67122. EcoRI digestionexcises the cDNA insert present in pMT2-VWF, yielding pMT2 in linearform which can be ligated and used to transform E. coli HB 101 or DH-5to ampicillin resistance. Plasmid pMT2 DNA can be prepared byconventional methods. pMT2 CXM is then constructed using loopout/inmutagenesis [Morinaga, et al., Biotechnology 84: 636 (1984). Thisremoves bases 1075 to 1145 relative to the Hind III site near the SV40origin of replication and enhancer sequences of pMT2. In addition itinserts a sequence containing the recognition site for the restrictionendonuclease Xho I. A derivative of pMT2CXM, termed pMT23, containsrecognition sites for the restriction endonucleases PstI, Eco RI, SalIand XhoI. Plasmid pMT2 CXM and pMT23 DNA may be prepared by conventionalmethods.

pEMC2β1 derived from pMT21 may also be suitable in practice of theinvention. pMT21 is derived from pMT2 which is derived from pMT2-VWF. Asdescribed above EcoRI digestion excises the cDNA insert present inpMT-VWF. yielding pMT2 in linear form which can be ligated and used totransform E. Coli HB 101 or DH-5 to ampicillin resistance. Plasmid pMT2DNA can be prepared by conventional methods.

pMT21 is derived from pMT2 through the following two modifications.First, 76 bp of the 5′ untranslated region of the DHFR cDNA including astretch of 19 G residues from G/C tailing for cDNA cloning is deleted.In this process, a XhoI site is inserted to obtain the followingsequence immediately upstream from DHFR. Second, a unique ClaI site isintroduced by digestion with ECoRV and XbaI, treatment with Klenowfragment of DNA polymerase I, and ligation to a ClaI linker (CATCGATG).This deletes a 250 bp segment from the adenovirus associated RNA (VAI)region but does not interfere with VAI RNA gene expression or function.pMT21 is digested with EcoRI and XhoI, and used to derive the vectorpEMC2B1.

A portion of the EMCV leader is obtained from pMT2-ECAT1 [S. K. Jung, etal, J. Virol 63:1651-1660 (1989)] by digestion with Eco RI and PstI,resulting in a 2752 bp fragment. This fragment is digested with TaqIyielding an Eco RI-TaqI fragment of 508 bp which is purified byelectrophoresis on low melting agarose gel. A 68 bp adapter and itscomplementary strand are synthesized with a 5′ TaqI protruding end and a3′ XhoI protruding end which has a sequence which matches the EMC virusleader sequence from nucleotide 763 to 827. It also changes the ATG atposition 10 within the EMC virus leader to an ATT and is followed by aXhoI site. A three way ligation of the pMT21 Eco RI-XhoI fragment, theEMC virus EcoRI-TaqI fragment, and the 68 bp oligonucleotide adapterTaqI-XhoI adapter resulting in the vector pEMC2β1.

This vector contains the SV40 origin of replication and enhancer, theadenovirus major late promoter, a cDNA copy of the majority of theadenovirus tripartite leader sequence, a small hybrid interveningsequence, an SV40 polyadenylation signal and the adenovirus VA I gene,DHFR and β-lactamase markers and an EMC sequence, in appropriaterelationships to direct the high level expression of the desired cDNA inmammalian cells.

The construction of vectors may involve modification of the BMP-12 DNAsequences. For instance, BMP-12 cDNA can be modified by removing thenon-coding nucleotides on the 5′ and 3′ ends of the coding region. Thedeleted non-coding coding nucleotides may or may not be replaced byother sequences known to be beneficial for expression. These vectors aretransformed into appropriate host cells for expression of BMP-12proteins. Additionally, the sequence of SEQ ID NO:1 or other sequencesencoding BMP-12 proteins can be manipulated to express BMP-12 protein byisolating the mature.coding sequence of nucleotides 571 to 882 of SEQ IDNO:1 and adding at the 5′ end sequences encoding the completepropeptides of other BMP proteins.

For example, one skilled in the art can make a fusion protein in whichthe propeptide of BMP-2 is linked in operable fashion to the matureBMP-12 peptide by preparing a DNA vector in which the DNA sequenceencoding the BMP-2 propeptide is linked in proper reading frame to theDNA sequence encoding the mature BMP-12 peptide. The DNA sequence ofsuch a fusion protein is shown in SEQUENCE ID NO:27.

One skilled in the art can manipulate the sequences of SEQ ID NO:1 byeliminating or replacing the mammalian regulatory sequences flanking thecoding sequence with bacterial sequences to create bacterial vectors forintracellular or extracellular expression by bacterial cells, asdescribed above. As another example, the coding sequences could befurther manipulated (e.g. ligated to other known linkers or modified bydeleting non-coding sequences therefrom or altering nucleotides thereinby other known techniques). The modified BMP-12 coding sequence couldthen be inserted into a known bacterial vector using procedures such asdescribed in T. Taniguchi et al., Proc. Natl Acad. Sci. USA,77:5230-5233 (1980). This exemplary bacterial vector could then betransformed into bacterial host cells and a BMP-12 protein expressedthereby. For a strategy for producing extracellular expression of BMP-12proteins in bacterial cells, see, e.g. European patent application EPA177,343.

Similar manipulations can be performed for the construction of an insectvector [See, e.g. procedures described in published European patentapplication 155,476] for expression in insect cells. A yeast vectorcould also be constructed employing yeast regulatory sequences forintracellular or extracellular expression of the factors of the presentinvention by yeast cells. [See, e.g., procedures described in publishedPCT application WO86/00639 and European patent application EPA 123,289].

A method for producing high levels of a BMP-12 protein of the inventionin mammalian cells may involve the construction of cells containingmultiple copies of the heterologous BMP-12 gene: The heterologous, geneis linked to an amplifiable marker, e.g. the dihydrofolate reductase(DHFR) gene for which cells containing increased gene copies can beselected for propagation in increasing concentrations of methotrexate(MTX) according to the procedures of Kaufman and Sharp, J. Mol. Biol.,159:601-629 (1982). This approach can be employed with a number ofdifferent cell types.

For example, a plasmid containing a DNA sequence for a BMP-12 of theinvention in operative association with other plasmid sequences enablingexpression thereof and the DHFR expression plasmid pAdA26SV(A)3 [Kaufmanand Sharp, Mol. Cell. Biol., 2:1304 (1982)] can be co-introduced intoDHFR-deficient CHO cells, DUKX-BII, by various methods including calciumphosphate coprecipitation and transfection, electroporation orprotoplast fusion. DHFR expressing transformants are selected for growthin alpha media with dialyzed fetal calf serum, and subsequently selectedfor amplification by growth in increasing concentrations of MTX (e.g.sequential steps in 0.02, 0.2, 1.0 and 5 uM MTX) as described in Kaufmanet al., Mol Cell Biol., 5:1750 (1983). Transformants are cloned, andbiologically active BMP-12 expression is monitored by the Rosen-modifiedSampath-Reddi rat assay described below in Example 5. BMP-12 expressionshould increase with increasing levels of MTX resistance. BMP-12polypeptides are characterized using standard techniques known in theart such as pulse labeling with [35S] methionine or cysteine andpolyacrylamide gel electrophoresis. Similar procedures can be followedto produce other related BMP-12 proteins.

EXAMPLE 3

Preparation of BMP-2 Propeptide/BMP-12 Mature Peptide Fusion

In order to construct a vector encoding the BMP-2 propeptide/BMP-12mature peptide fusion, the following cloning procedure was used to fusethe two sequences together.

First, a DNA restriction enzyme fragment comprising the propeptide ofhuman BMP-2 protein, comprising nucleotides 1 through 843 of SEQ IDNO:27 is cut from pBMP2ΔEMC. pBMP2ΔEMC is a plasmid derived from lambdaU20S-39 (ATCC #40345) comprising the entire coding sequence for humanBMP-2 protein with the non-translated 5′ and 3′ sequences of BMP-2deleted from the vector. The 5′ restriction enzyme used was Bgl II andit cuts pBMP2ΔEMC in the vector at nucleotide 979. The 3′ restrictionenzyme used was Mae II and it cuts pBMP2ΔEMC in the BMP-2 propeptide atnucleotide 1925, just short of the carboxy terminus. The resulting 954base pair product was then gel isolated and gene cleaned. Second, a DNArestriction enzyme fragment comprising the 5′ portion of the humanBMP-12 mature peptide DNA sequence, is cut from pPCR1-1#2 V1-1 (ATCC#69517). The 5′ restriction enzyme used was Eae I and it cuts pPCR1-1#2V1-1 just 3′ of N-terminus of the human BMP-12 mature peptide sequence.The resulting 259 base pair product was gel isolated and gene cleaned.Third, two DNA oligos were designed and synthesized, so that whenannealed would form a tiny DNA fragment comprising fusion sequence ofthe extreme 3′ end of the human BMP-2 propeptide and the 5′ end ofBMP-12 mature peptide. The DNA fragment has a 5′ Mae II complimentarysticky end which anneals to the 3′ restriction enzyme fragmentcomprising the human BMP-2 propeptide. The annealed oligo DNA fragmenthas a 3′ Eae I complimentary sticky end which anneals to the 5′ of therestriction enzyme fragment comprising the mature peptide of humanBMP-12. The coding strand oligo is named B2/12 and is 13 base pairslong. Next, a DNA fragment encoding the 123 base pairs at the 3′ end ofthe BMP-12 mature peptide fragment was obtained as follows. First, a DNAfragment comprising the propeptide of human BMP-2 protein, comprisingnucleotides 1 through 846 is PCR amplified. from pBMP2ΔEMC. The 5′primer (oligo 655a) anneals just 5′ of the polylinker. The 3′ primer(BMPpro3) anneals to the BMP-2 propeptide 3′ end and introduces a Bgl IIrestriction enzyme site by silent sequence mutations. The resulting PCRproduct was cut with Sal 1, which cleaves in the polylinker, and Bgl II.The 850 base pair restriction enzyme fragment (ending in amino acidsequence REKR) was gel isolated and gene cleaned. The BMP-12 maturepeptide was PCR amplified using a 5′ primer (oligo 5-1) encoding the BglII restriction enzyme site by silent sequence mutations, and annealingto the 5′ end of a possible mature cleavage product, beginning withamino acid sequence SRCS. The 3′ primer (V1-1 3) anneals to the BMP-12mature peptide 3′ end and introduces a Xba I restriction enzyme siteafter the stop codon. The resulting PCR product was cut with Bgl II andXba I. The 321 base pair, restriction enzyme fragment was gel isolatedand gene cleaned.

The two restriction fragments were three-way ligated into a previouslySalI and XbaI cut vector. The resultant construct was sequenced to checkfor PCR induced errors and a silent C to T mutation was observed at basepair 185 in the propeptide. This plasmid was designated pREKRSRC. ThenpREKRSRC was cut with BglII and NgoMI, and the vector fragmentencompassing the last 123 base pairs of the BMP12 mature sequence wasthereby isolated. The three restriction fragments and the annealedoligolinker were four-way ligated to yield pREKR-TAL with the BMP-2propeptide with the mature cleavage site at the 3′ end fused to the(TAL) 5′ end of the BMP-12 mature peptide. The coding sequence of theresulting ligated vector is shown in SEQ ID NO:27.

EXAMPLE 4

Biological Activity of Expressed BMP-12

To measure the biological activity of the expressed BMP-12 proteinsobtained in Example 2 above, the proteins are recovered from the cellculture and purified by isolating the BMP-12 proteins from otherproteinaceous materials with which they are co-produced as well as fromother contaminants. The purified protein may be assayed in accordancewith the rat assay described below in Example 5.

Purification is carried out using standard techniques known to thoseskilled in the art.

Protein analysis is conducted using standard techniques such as SDS-PAGEacrylamide [Laemmli, Nature 227:680 (1970)] stained with Coomassie Blueor silver [Oakley, et al. Anal. Biochem. 105:361 (1980)] and byimmunoblot [Towbin, et al. Proc. Natl. Acad. Sci. USA 76:4350 (1979)]

EXAMPLE 5

Rosen Modified Sampath-Reddi Assay

A modified version of the rat ectopic implant assay described in Sampathand Reddi, Proc. Natl. Acad. Sci. USA, 80:6591-6595 (1983) is used toevaluate the activity of the BMP-12 proteins. This modified assay isherein called the Rosen-modified Sampath-Reddi assay. The assay has beenwidely used to evaluate the bone and cartilage-inducing activity ofBMPs. The ethanol precipitation step of the Sampath-Reddi procedure isreplaced by dialyzing (if the composition is a solution) or diafiltering(if the composition is a suspension the fraction to be assayed againstwater. The solution or suspension is then equilibrated to 0.1% TFA. Theresulting solution is added to 20 mg of rat matrix. A mock rat matrixsample not treated with the protein serves as a control. This materialis frozen and lyophilized and the resulting powder enclosed in #5gelatin capsules. The capsules are implanted subcutaneously in theabdominal thoracic area of 21-49 day old male Long Evans rats. Theimplants are removed after 10 days. A section of each implant is fixedand processed for histological analysis. 1 μm glycolmethacrylatesections are stained with Von Kossa and acid fuschin to score the amountof induced tendon/ligament-like tissue formation present in eachimplant.

BMP-12 was implanted in the rats in doses of 1, 5, 25 and 50 μg perimplant for 10 days. BMP-2 at a dose of 5 μg was included as a positivecontrol. For all doses of BMP-12 tested, no bone or cartilage formationwas observed in the implants after ten days. Instead, the implants werefilled with tissue resembling embryonic tendon, which is easilyrecognized by the presence of dense bundles of fibroblasts oriented inthe same plane and packed tightly together. [Tendon/ligament-like tissueis described, for example, in Ham and Cormack, Histology (JB LippincottCo). (1979), pp. 367-369, the disclosure of which is hereby incorporatedby reference] These findings were reproduced in a second set of assaysin which tendon/ligament-like tissues was present in all BMP-12containing implants. In contrast, the BMP-2 implants, as expected,showed cartilage and bone formation, but contained notendon/ligament-like tissue.

The BMP-12 proteins and related proteins of this invention may beassessed for activity on this assay.

EXAMPLE 6

Using methods in accordance with the above examples. with minormodifications within the skill of the art, human MP52 protein and themurine homologue of BMP-13 protein were expressed and assayed fortendon/ligament-like tissue inducing activity. All proteins showedcomparable results, similar to those described above for human BMP-12.

The foregoing descriptions detail presently preferred embodiments of thepresent invention. Numerous modifications and variations in practicethereof are expected to occur to those skilled in the,art uponconsideration of these descriptions. Those modifications and variationsare believed to be encompassed within the claims appended hereto. Thedisclosure of all references discussed herein are hereby incorporated byreference.

1-28. (canceled)
 29. A method for regenerating a functional attachmentbetween tendon and/or ligament tissue and bone comprising administeringan effective amount of a protein selected from the group consisting ofBMP-12, BMP-13, and MP-52 to a patient in need of same.
 30. The methodof claim 29, wherein the protein administered is BMP-12.
 31. The methodof claim 29, wherein the protein administered is BMP-13.
 32. The methodof claim 29, wherein the protein administered is MP-52.
 33. A method fortreating a tendon and/or ligament defect comprising administering aneffective amount of a protein selected from the group consisting ofBMP-12, BMP-13, and MP-52 to a patient in need of same.
 34. The methodof claim 33, wherein the protein administered is BMP-12.
 35. The methodof claim 33, wherein the protein administered is BMP-13.
 36. The methodof claim 33, wherein the protein administered is MP-52.